ic 54: collagen cross-linking. indications, applications ...€¦ · - cornea collagen...

17 Tsocha Str., 115 21 Athens, Greece Phone: +30 210 7472 777 – Fax: +30 210 7472 789 – Emergency phone: +30 6945993598

email: [email protected], www.laservision.gr

ESCRS, Copenhagen 2016

IC 54: Collagen Cross-Linking. Indications, Applications, results, complications and evolving technology

Senior Instructor: A. John Kanellopoulos, MD

Co- authors: B. Armstrong, MD R. Pineda, MD S. Jacob, MD D. Stulting, MD G. Pamel, MD

Course Description:

Synopsis: Didactic approach to the management of progressive cornea ectasia associated with keratoconus and refractive surgery. Several surgical treatment modalities utilized internationally will be presented, including: collagen cross-linking with ultraviolet radiation A in order to halt ectasia, combined in some cases with a customised excimer laser ablation to facilitate visual rehabilitation (as presented in previous ESCRS meetings by the author), these alternatives to Intracornea ring segment implantation, lamelllar grafts as well as penetrating graft techniques will be analyzed. Surgical and medical treatment technique, indications, potential complications and their management as well as clinical experience pearls will be presented Objective: The participants will share our vast experience in managing prograssive keratoconus and post-LASIK ectasia in order to visually rehabilitate these patients. Pearls on indications, patient selection, surgical technique and complication management for safe and effective results will be presented and discussed with the participants. Outline: 1-Keratoconus surgical management, Literature review: -Thermokeratoplasty - Lamellar grafts - INTACS - Penetrating keratoplasty - Cornea Collagen Cross-linking 2-Post-LASIK ectasia-surgical management, literature review:

A. John Kanellopoulos, MD Clinical Professor Of Ophthalmology, NYU Cornea Transplantation and Refractive Surgery

17 Tsocha Str., 115 21 Athens, Greece Phone: +30 210 7472 777 – Fax: +30 210 7472 789 – Emergency phone: +30 6945993598

email: [email protected], www.laservision.gr

-How to avoid-risk factors -Thermokeratoplasty - Lamellar grafts - INTACS - Penetrating keratoplasty - Cornea Collagen Cross-linking 3-patient selection a)- Indications b)-medical contraindications c)-pre-operative evaluation and refractive error 4-Stabilization of ectasia: -INTACS -Collagen cross-linking -lamellar tissue support 5-Collagen cross-linking: -energy source, luminance and duration -protective riboflavin A -cornea pachymetry issues -topographic and elevation changes -stabilization of ectasia -When is it best to intervene? -FDA issues 6-Customised enhancement techniques a) wavefront-guided b)Topography-guided c)Asphericity adjustment 7- Microkeratome-assisted lamellar keratplasty technique a)- basic principles b)-pre-operative evaluation parametres c)-Surgical technique d)-Possible complications and their management e)-Clinical data and review of the literature 8- Penetrating keratoplasty considerations: a)- basic principles b)-pre-operative evaluation parametres c)-Surgical technique d)-Possible complications and their management e)-Clinical data and review of the literature 9- Refractive surgery enhancements following these procedures 10-surgery in action Step-by-step action on several procedures on tape, question-answer session and coverage of basic problem-shooting with the panelists

342 Copyright © SLACK Incorporated

O R I G I N A L A R T I C L E

Corneal Refractive Power and Symmetry Changes Following Normalization of Ectasias Treated With Partial Topography-Guided PTK Combined With Higher-Fluence CXL (The Athens Protocol)Anastasios John Kanellopoulos, MD; George Asimellis, PhD

ABSTRACT

PURPOSE: To investigate preoperative and postopera-tive anterior and posterior keratometry and simulated corneal astigmatism in keratoconic eyes treated with collagen cross-linking combined with anterior surface normalization by partial topography-guided excimer ab-lation (the Athens Protocol).

METHODS: Anterior and posterior corneal keratom-etry were measured by Scheimpflug imaging for 267 untreated keratoconic eyes. Following treatment, they were assessed 1 year postoperatively.

RESULTS: Before treatment, average anterior kerato-metric value was 47.06 ± 6.02 diopters (D) for flat and 51.24 ± 6.75 D for steep. The posterior kerato-metric values were -6.70 ± 0.99 D (flat) and -7.67 ± 1.15 D (steep). Anterior astigmatism was on aver-age with-the-rule (-1.97 ± 6.21 D), whereas posterior astigmatism was against-the-rule (+0.53 ± 1.02 D). The posterior and anterior astigmatism were highly cor-related (r2 = 0.839). After treatment, anterior kerato-metric values were 43.97 ± 5.81 D (flat) and 46.55 ± 6.82 D (steep). Posterior keratometric values were -6.58 ± 1.05 D (flat) and -7.69 ± 1.22 D (steep). An-terior astigmatism was on average with-the-rule (-1.56 ± 3.80 D), whereas posterior astigmatism was against-the-rule (+0.45 ± 1.29 D). The statistically significant (P < .05) keratometric changes indicated anterior sur-face flattening -3.09 ± 2.67 D (flat) and -4.19 ± 2.96 D (steep). The posterior keratometric changes were not statistically significant (P > .05).

CONCLUSIONS: Before treatment, there was a strong correlation between posterior and anterior corneal astig-matism. After treatment, statistically significant anterior keratometric values flattened. The posterior surface keratometric values did not demonstrate statistically significant postoperative change: there was minimal posterior change, despite the significant anterior sur-face normalization.

[J Refract Surg. 2014;30(5):342-346.]

eratoconus assessment employs indicators such as keratometric values, inferior-superior index, skew percentage, astigmatism, and the KISA% index.1

Acceptable quantitative keratometric criteria include cen-tral corneal refractive power larger than 47.2 diopters (D), inferior-superior dioptric asymmetry larger than 1.2 D, and simulated astigmatism, expressed as the difference between steep and flat keratometric values greater than 1.5 D.2 The steep and flat meridian keratometric values correspond to the smaller and larger anterior corneal curvature radius, respectively.

Corneal cross-linking (CXL) is an in vivo intrastromal pho-to-oxidative technique with riboflavin and ultraviolet-A light aiming to address the advancing corneal ectasia and, conse-quently, the keratoconus progression. With CXL, additional covalent bonding between stromal collagen can be achieved, which stabilizes the collagen framework structure.3 The re-modeling effects of CXL on the cornea can be described by the reduction of mean anterior surface keratometric values.4 Few studies have been published on the quantitative link be-tween anterior and posterior keratometric values in kerato-conic eyes or particularly on the postoperative effects of CXL on either corneal surface.

This study aims to investigate the distribution of and rela-tionship between anterior and posterior corneal keratometric values and simulated anterior and posterior astigmatism on a large group of clinically diagnosed, untreated keratoconic eyes, and the 1-year postoperative effects on both anterior and posterior keratometric values and astigmatism induced by a combined procedure known as the Athens Protocol,5,6 which intends to arrest the keratoconus progression and nor-malize the anterior corneal surface.

From Laservision.gr Eye Institute, Athens, Greece (AJK, GA); and New York University School of Medicine, New York, New York (AJK).

Submitted: July 22, 2013; Accepted: January 16, 2014; Posted online: May 2, 2014

The authors have no financial or proprietary interest in the materials pre-sented herein.

Correspondence: Anastasios John Kanellopoulos, MD, Laservision.gr Eye Institute, 17 Tsocha str, Athens 11521, Greece. E-mail: [email protected]

doi:10.3928/1081597X-20140416-03

K

343Journal of Refractive Surgery

Corneal Refractive Power and Symmetry Changes/Kanellopoulos & Asimellis

PATIENTS AND METHODSThis study was performed in patients visiting our

clinical practice and which received approval by the ethics committee of our institution and adhered to the tenets of the Declaration of Helsinki. Informed written consent was obtained from all patients at the time of the first clinical visit.

INCLUSION CRITERIA AND SURGICAL TECHNIQUEThe study group consisted of 267 eyes. Patients’ ages

at the time of the screening ranged from 19 to 57 years. Each patient received a complete ocular examination, including subjective refraction, visual acuity, and slit-lamp biomicroscopy for clinical signs of keratoconus. Inclusion criteria included definite findings consistent with keratoconus, described by the Collaborative Lon-gitudinal Evaluation of Keratoconus group.7 Exclusion criteria included systemic disease, previous corneal surgery, history of chemical injury or delayed epitheli-al healing, and pregnancy or lactation (female patients) during the study.

The same cases received treatment with the Athens Protocol,8 which involved excimer laser epithelial de-bridement (50 µm), partial (maximum ablation 80 µm) topography-guided excimer laser stromal ablation, and high-fluence ultraviolet-A irradiation (10 mW/cm2), accelerated (10’) CXL performed with the Avedro KXL System (Avedro, Inc., Waltham, MA). The Athens Pro-tocol treatments employed the Oculyzer II (WaveLight AG, Erlagen, Germany)9,10 for corneal topography im-aging. The Oculyzer II is a high-resolution Pentacam Scheimpflug imaging rotating camera (Oculus Optik-geräte GmbH, Wetzlar, Germany) incorporated by a proprietary network in the WaveLight Refractive Suite (WaveLight AG)11 to provide corneal elevation data to the excimer laser, namely the EX500 (Alcon Laborato-ries, Fort Worth, TX).

For a patient to be considered for the Athens Proto-col procedure, the criteria included clinical diagnosis of progressive keratoconus, minimum age of 18 years, and 300-µm minimum corneal thickness. Further de-tails of the Athens Protocol procedure have been pub-lished.12,13 All patients were observed at 1 week and 1, 3, 6, and 12 months postoperatively, and on an annual basis thereafter. The corneal data reported in this study after treatment comprised measurements obtained at the 12-month postoperative visit.

IMAGING, MEASUREMENT, AND ANALYSISThe Scheimpflug camera (Oculyzer II) was regularly

calibrated according to manufacturer recommenda-tions. The measurements were obtained and processed via the Examination Software (Version 1.17r47; Wave-

Light AG). The default settings and 25 images per sin-gle acquisition were implemented. Keratometric and simulated astigmatism data were obtained with the best fit toric ellipsoid reference surface.

Linear regression analysis was performed to seek possible correlations. Descriptive and comparative sta-tistics and analysis of variance were performed with statistics tools provided by Minitab version 16.2.3. (MiniTab Ltd., Coventry, UK) and Origin Lab version 9 (OriginLab Corp, Northampton, MA). A P value less than .05 was considered statistically significant.

RESULTSThe sample consisted of 267 eyes (82 female and

185 male). There was preponderance toward male gen-der, consistent with our clinical experience in male-to-female incidence in keratoconic patients14 and keratoconus incidence studies.15 Of the 267 eyes, 140 were right eyes and 127 were left eyes. The average age for all patients at the time of the procedure was 30.80 ± 7.25 years (range: 19 to 57 years). The aver-age preoperative corrected distance visual acuity was 20/32, ranging from 20/200 to 20/20 (0.628 ± 0.241). One year postoperatively, average corrected distance visual acuity was 20/25, ranging from 20/100 to 20/16 (0.762 ± 0.225).

ANTERIOR AND POSTERIOR KERATOMETRY AND CORNEAL ASTIGMATISM BEFORE TREATMENT

Average, standard deviation, and maximum and minimum anterior and posterior central corneal sur-face keratometric values before and after treatment with the Athens Protocol are reported in Table 1.

The astigmatism sign is dependent on the flat me-ridian horizontal or vertical orientation: the sign is considered positive if the flat meridian orientation is between +45° and +135°, which is called “against-the-rule,” whereas it is considered negative if between -45° and +45°, which is called “with-the-rule.”

After treatment, the resulting anterior astigmatism was on average with-the-rule, whereas the posterior astigmatism was against-the-rule (Figure 1). Paired analysis of the differences regarding astigmatism change indicated a P value of .067 for the anterior sur-face and .358 for the posterior surface.

KERATOMETRIC CHANGES AND ANALYSISFigure 1 illustrates the correlation between poste-

rior versus anterior astigmatism in the form of scat-ter and fitted line plots with 95% confidence intervals and 95% prediction intervals before and after treat-ment. Before treatment, the coefficient of determina-tion (r2) was 0.839 with a P value less than .001. After



treatment, r2 was 0.407 and P value less than .001 (Table 2).

Data analysis indicates that the mean of the paired differences regarding the flat anterior keratometric val-

ues was reduced (flattened) postoperatively by -3.09 ± 2.69 D, or -6.56%, and was statistically significant (P < .05) (Table 2). The uncertainty associated with esti-mating the difference from sample data indicated, with a 95% confidence interval, that the true difference was between -2.76 and -3.41 D. The steep anterior keratomet-ric values showed a postoperative flattening by -4.19 ± 2.96 D, or -8.19%, again statistically significant (P < .05). The true 95% difference was between -3.84 and -4.55 D.

The mean of the paired differences regarding the flat posterior keratometric values showed an increase of +0.12 ± 0.61 D, or -1.76% (considering the negative sign of the posterior keratometric values). The 95% true difference was between -0.191 and -0.0449 D. The analysis for the steep posterior keratometric val-ues showed a postoperative change of -0.02 ± 0.55 D or +0.04% (95% true difference between -0.0485 and +0.0829 D). These differences were not statistically significant (flat P = .135, steep P = .606).

DISCUSSIONIn this study, a rotating Scheimpflug camera was

employed to measure both the anterior and posterior corneal curvature in a large number (267) of kerato-conic cases, before and after (1 year postoperatively) a combined CXL and anterior surface excimer laser nor-malization. In the case of keratoconus, highly irregu-lar keratometric values were present. For example, the simultaneous investigation of anterior and posterior corneal keratometric values has indicated statistically significant differences between normal and keratoco-nus-suspect eyes.16 In a study evaluating keratometric values in keratoconic compared to normal eyes, the

TABLE 1Anterior and Posterior Corneal Surface Keratometry and Astigmatism as Measured in the 8-mm Diameter Zone Before and After Treatment

Before Treatment After Treatment

Parameter Average (D) SD (D) Max (D) Min (D) Average (D) SD (D) Max (D) Min (D)

Anterior cornea

Flat 47.06 ±6.02 78.50 33.70 43.97 ±5.81 73.2 30.1

Steep 51.24 ±6.75 80.70 39.50 47.04 ±6.86 81.3 33.9

Mean 49.03 ±6.21 78.80 38.80 46.37 ±6.73 80.9 31.9

Astigmatism -1.97 ±6.21 11.30 -12.40 -1.56 ±3.80 12.4 -11.5

Posterior cornea

Flat -6.70 ±0.99 -4.60 -9.90 -6.58 ±1.05 -3.3 -10.4

Steep -7.67 ±1.15 -5.60 -11.00 -7.69 ±1.22 -5.2 -13.2

Mean -7.08 ±1.40 8.50 -10.20 -7.08 ±1.06 -4.2 -11.5

Astigmatism +0.53 ±1.02 +4.00 -2.60 +0.45 ±1.29 +4.3 -5.3

SD = standard deviation; D = diopters; Max = maximum; Min = minimum

Figure 1. Scatter and fitted line plots of posterior astigmatism expressed in diopters (D) versus anterior astigmatism (also expressed in D) with 95% confidence intervals (CI) and 95% prediction intervals (PI). (Top) Before and (bottom) after treatment.



mean value was 43.28 ± 1.17 D (range: 41.53 to 45.40 D) for normal eyes and 49.29 ± 4.37 D (range: 42.97 to 60.33 D) for keratoconic eyes.17

In our study, the average keratometric values be-fore treatment were 47.06 ± 6.02 D (range: 33.7 to 78.5 D) for flat and 51.24 ± 6.75 D (range: 39.5 to 80.7 D) for steep. As shown in the corresponding box plots in Figures A-C (available in the online version of this article), 95% of the sample population had a steep keratometric value greater than 46.025 D, consistent with the Collaborative Longitudinal Evaluation of Ker-atoconus group standards.7

In the current study, before treatment the results were characterized by a pattern of linear correlation between anterior and posterior astigmatism, as shown in the fitted line plot of Figure 1. The anterior astig-matism before treatment is generally with-the-rule and the posterior astigmatism is against-the-rule, with a linear fit coefficient ratio of -0.2067 and a robust coef-ficient of determination (r2) of 0.839. Thus, posterior corneal keratometric values appeared in this large ker-atoconic population to be associated with the anterior keratometric values.

However, this pattern does not seem to be consistent after treatment (Figure 1). Although the ratio was simi-lar (-0.26), the coefficient of determination was con-sidered poor (r2 = 0.405). Our results indicate that this was due to the induced changes in the anterior surface. Specifically, there was a statistically significant reduc-tion in the anterior keratometric values after the Ath-ens Protocol procedure demonstrated by a flattening of -3.09 D for the flat and -4.19 D for the steep merid-ians, in agreement with other studies of keratoconic cases receiving CXL treatment.18 The observed changes in the posterior keratometric values were not statisti-cally significant, and thus no measurable change in the posterior corneal surface could be established in our study.

The issue of posterior surface change following CXL,19 surface ablation,20 or a combination of both pro-cedures has to be viewed with skepticism. We present these data with the reservation that measurements of the posterior surface might be influenced by the al-gorithm involving corneal thickness measurements, which, in the case of the densitometry principle-based imaging, might be influenced in the CXL-treated cor-neas.21,22 Scheimpflug imaging in its current form can only provide data for the posterior surface on clear, normal corneas. Any significant cornea irregularity or light conduction interference may bias the findings. Also possible is that the denser CXL effect observed with application of the Athens Protocol may mask pos-terior surface changes. Therefore, although we observed

some posterior surface changes, as expected, due to the dramatic alteration of the anterior surface and cornea stromal changes, these appear to be non-significant in comparison to the anterior surface changes.

There is an obvious clinical dilemma in deciding to ablate thin corneas with ectasia. Traditional clinical experience in the pro-CXL era may consider this dan-gerous for the ectasia progression. We investigated the principle of posterior curvature stability in these eyes because of a critical factor of establishing the safety of ablating these corneas. As expected, the posterior sur-face keratometric values did not show statistically sig-nificant postoperative change. Various data suggest that there is minimal change in the biomechanical behavior of the ectatic cornea after the Athens Protocol, despite the dramatic change in the anterior surface cone.

Before treatment the elevated anterior and posterior keratometric values were highly correlated. After treat-ment showed statistically significant anterior surface flattening, consistent with our previous studies. The posterior surface did not show a statistically signifi-cant change, validating the accomplished cornea sta-bility despite the interventional thinning.

AUTHOR CONTRIBUTIONSConception and design (AJK); data collection (AJK); analysis and

interpretation of data (AJK, GA); writing the manuscript (GA); criti-cal revision of the manuscript (AJK, GA)

REFERENCES 1. Rabinowitz YS, Rasheed K. KISA% index: a quantitative vid-

eokeratography algorithm embodying minimal topographic criteria for diagnosing keratoconus. J Cataract Refract Surg. 1999;25:1327-1335.

TABLE 2

Anterior and Posterior Corneal Surface Keratometry Changes Before

and After Treatmenta

ParameterK1 Anterior

(D)K2 Anterior

(D)K1 Posterior

(D)K2 Posterior

(D)

Relative change (%) -6.56 -8.19 -1.76 -0.04

Average change -3.09 -4.19 0.12 -0.02

SD 2.67 2.96 0.61 0.55

Max 4.95 1.70 3.60 2.00

Min -17.30 -17.30 -1.50 -2.90

Pb < .01 < .01 .135 .606

K = keratometry; K1 = flat; K2 = steep; D = diopters; SD = standard deviation; Max = maximum; Min = minimum aAverage change is defined as the postoperative (after treatment) minus the preop-erative value (before treatment), computed on each individual. Relative change is defined as the percent of the average change of the parameter with regard to the respective preoperative value. bTwo sample t test.



2. Rabinowitz YS. Videokeratographic indices to aid in screening for keratoconus. J Refract Surg. 1995;11:371-379.

3. Wollensak G. Crosslinking treatment of progressive keratoco-nus: a new hope. Curr Opin Ophthalmol. 2006;17:356-360.

4. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen cross-linking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg. 2008;34:796-801.

5. Kanellopoulos AJ, Binder PS. Management of corneal ectasia after LASIK with combined, same-day, topography-guided par-tial transepithelial PRK and collagen cross-linking: the Athens Protocol. J Refract Surg. 2011;27:323-331.

6. Kanellopoulos AJ, Asimellis G. Comparison of Placido disc and Scheimpflug image-derived topography-guided excimer laser surface normalization combined with higher fluence CXL: the Athens Protocol, in progressive keratoconus. Clin Ophthalmol. 2013;7:1385-1396.

7. Zadnik K, Barr JT, Edrington TB, et al. Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. Invest Ophthalmol Vis Sci. 1998;39:2537-2546.

8. Kanellopoulos AJ. Long term results of a prospective random-ized bilateral eye comparison trial of higher fluence, short-er duration ultraviolet A radiation, and riboflavin collagen cross linking for progressive keratoconus. Clin Ophthalmol. 2012;6:97-101.

9. Kanellopoulos AJ, Asimellis G. Correlation between central corneal thickness, anterior chamber depth, and corneal kera-tometric values as measured by Oculyzer II and WaveLight OB820 in preoperative cataract surgery patients. J Refract Surg. 2012;28:895-900.

10. McAlinden C, Khadka J, Pesudovs K. A comprehensive evalu-ation of the precision (repeatability and reproducibility) of the Oculus Pentacam HR. Invest Ophthalmol Vis Sci. 2011;52:7731-7737.

11. Kanellopoulos AJ, Asimellis G. Long-term bladeless LASIK out-comes with the FS200 femtosecond and EX500 Excimer Laser workstation: the Refractive Suite. Clin Ophthalmol. 2013;7:261-269.

12. Kanellopoulos AJ. Comparison of sequential vs same-day simul-taneous collagen cross-linking and topography-guided PRK for treatment of keratoconus. J Refract Surg. 2009;25:S812-S818.

13. Kanellopoulos AJ. Long term results of a prospective random-ized bilateral eye comparison trial of higher fluence, short-er duration ultraviolet A radiation, and riboflavin collagen cross linking for progressive keratoconus. Clin Ophthalmol. 2012;6:97-101.

14. Kanellopoulos AJ, Asimellis G. Introduction of quantitative and qualitative cornea optical coherence tomography findings induced by collagen cross-linking for keratoconus: a novel ef-fect measurement benchmark. Clin Ophthalmol. 2013;7:329-335.

15. Gordon-Shaag A, Millodot M, Shneor E. The epidemiology and etiology of keratoconus. Int J Keratoco Ectatic Corneal Dis. 2012;1:7-15.

16. Schlegel Z, Hoang-Xuan T, Gatinel D. Comparison of and cor-relation between anterior and posterior corneal elevation maps in normal eyes and keratoconus-suspect eyes. J Cataract Refract Surg. 2008;34:789-795.

17. Alió JL, Shabayek MH. Corneal higher order aberrations: a method to grade keratoconus. J Refract Surg. 2006;22:539-545.

18. Mazzotta C, Caporossi T, Denaro R, et al. Morphological and functional correlations in riboflavin UV A corneal collagen cross-linking for keratoconus. Acta Ophthalmol. 2012;90:259-265.

19. Kránitz K, Kovács I, Miháltz K, et al. Corneal changes in pro-gressive keratoconus after cross-linking assessed by Scheimp-flug camera. J Refract Surg. 2012;28:645-649.

20. Sy ME, Ramirez-Miranda A, Zarei-Ghanavati S, Engle J, Danesh J, Hamilton DR. Comparison of posterior corneal imaging be-fore and after LASIK using dual rotating scheimpflug and scan-ning slit-beam corneal tomography systems. J Refract Surg. 2013;29:96-101.

21. Kanellopoulos AJ, Asimellis G. Keratoconus management: long-term stability of topography-guided normalization com-bined with high-fluence CXL stabilization (The Athens Proto-col). J Refract Surg. 2014;30:88-92.

22. Nakagawa T, Maeda N, Kosaki R, et al. Higher-order aberrations due to the posterior corneal surface in patients with keratoco-nus. Invest Ophthalmol Vis Sci. 2009;50:2660-2665.

Figure A. Anterior and posterior surface keratometric values (K1 [flat], K2 [steep], and Km [mean]) before treatment. (A, B) Box plots showing median level (indicated by ⊗, average symbol ⊕), 95% median confidence range box (black line boxes), and interquartile intervals range box (red line boxes). (C, D) The corresponding histogram plots )for K1 and K2. All units in keratometric diopters (D).

A B

C D

Figure B. Corneal astigmatism for the (left) before and (right) after treatment. (A, B) Box plots showing anterior and posterior astigmatism median level (indicated by ⊗, average symbol ⊕), 95% median confidence range box (black line boxes), and interquartile intervals range box (red line boxes). (C, D) The corresponding histogram plots for anterior and posterior astigmatism. All units in diopters (D).

A B

C D

Figure C. Anterior and posterior surface keratometric values (K1 [flat], K2 [steep], and Km [mean]) after treatment. (A, B) Box plots showing median level (indicated by ⊗, average symbol ⊕), 95% median confidence range box (black line boxes), and interquartile intervals range box (red line boxes). (C, D) The corresponding histogram plots for K1 and K2. All units in keratometric diopters (D).

A B

C D

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O R I G I N A L A R T I C L E

eratoconus is a degenerative bilateral, noninflam-matory disorder characterized by ectasia, thinning, and irregular corneal topography.1 The disorder

usually has onset at puberty and often progresses until the third decade of life, may manifest asymmetrically in the two eyes of the same patient, and can present with unpredictable visual acuity, particularly in relation to corneal irregularities.2 One of the acceptable options3 for progressive keratoconus management is corneal collagen cross-linking (CXL) with ri-boflavin and ultraviolet-A.4

To further improve the topographic and refractive out-comes, CXL can be combined with customized anterior sur-face normalization.5-7 Our team has developed a procedure8,9 we have termed the Athens Protocol,10 involving sequentially excimer laser epithelial debridement (50 µm), partial topog-raphy-guided excimer laser stromal ablation, and high-flu-ence ultraviolet-A irradiation (10 mW/cm2), accelerated (10’, or minutes) CXL. Early results11 and anterior segment optical coherence tomography quantitative findings12 are indicative of the long-term stability of the procedure.

Detailed studies on postoperative visual rehabilitation and anterior surface topographic changes by such combined CXL procedures are rare,13-16 particularly those reporting results longer than 1 year. This study aims to investigate safety and efficacy of the Athens Protocol procedure by analysis of long-term (3-year) refractive, topographic, pachymetric, and visual rehabilitation changes on clinical keratoconus management with the Athens Protocol in a large number of cases.

PATIENTS AND METHODSThis clinical study received approval by the Ethics Commit-

tee of our Institution and adhered to the tenets of the Declaration

KABSTRACT

PURPOSE: To investigate refractive, topometric, pachy-metric, and visual rehabilitation changes induced by an-terior surface normalization for keratoconus by partial topography-guided excimer laser ablation in conjunction with accelerated, high-fluence cross-linking.

METHODS: Two hundred thirty-one keratoconic cases subjected to the Athens Protocol procedure were stud-ied for visual acuity, keratometry, pachymetry, and an-terior surface irregularity indices up to 3 years postop-eratively by Scheimpflug imaging (Oculus Optikgeräte GmbH, Wetzlar, Germany).

RESULTS: Mean visual acuity changes at 3 years postoperatively were +0.38 ± 0.31 (range: -0.34 to +1.10) for uncorrected distance visual acuity and +0.20 ± 0.21 (range: -0.32 to +0.90) for corrected distance visual acuity. Mean K1 (flat meridian) kerato-metric values were 46.56 ± 3.83 diopters (D) (range: 39.75 to 58.30 D) preoperatively, 44.44 ± 3.97 D (range: 36.10 to 55.50 D) 1 month postoperatively, and 43.22 ± 3.80 D (range: 36.00 to 53.70 D) up to 3 years postoperatively. The average Index of Surface Variance was 98.48 ± 43.47 (range: 17 to 208) pre-operatively and 76.80 ± 38.41 (range: 7 to 190) up to 3 years postoperatively. The average Index of Height Decentration was 0.091 ± 0.053 µm (range: 0.006 to 0.275 µm) preoperatively and 0.057 ± 0.040 µm (range: 0.001 to 0.208 µm) up to 3 years postopera-tively. Mean thinnest corneal thickness was 451.91 ± 40.02 µm (range: 297 to 547 µm) preoperatively, 353.95 ± 53.90 µm (range: 196 to 480 µm) 1 month postoperatively, and 370.52 ± 58.21 µm (range: 218 to 500 µm) up to 3 years postoperatively.

CONCLUSIONS: The Athens Protocol to arrest keratec-tasia progression and improve corneal regularity dem-onstrates safe and effective results as a keratoconus management option. Progressive potential for long-term flattening validates using caution in the surface normal-ization to avoid overcorrection.

[J Refract Surg. 2014;30(2):88-92.]

From Laservision.gr Eye Institute, Athens, Greece (AJK, GA); and New York University School of Medicine, New York, New York (AJK).

Submitted: April 6, 2013; Accepted: August 21, 2013; Posted online: January 31, 2014

Dr. Kanellopoulos is a consultant for Alcon/WaveLight. The remaining author has no financial or proprietary interest in the materials presented herein.

Correspondence: Anastasios John Kanellopoulos, MD, 17 Tsocha str. Athens, Greece Postal Code 11521. E-mail: [email protected]

doi:10.3928/1081597X-20140120-03

Keratoconus Management: Long-Term Stability of Topography-Guided Normalization Combined With High-Fluence CXL Stabilization (The Athens Protocol)Anastasios John Kanellopoulos, MD; George Asimellis, PhD


The Athens Protocol/Kanellopoulos & Asimellis

of Helsinki. Written informed consent was obtained from each participant at the time of the intervention or the first clinical visit.

PATIENT INCLUSION CRITERIATwo hundred thirty-one consecutive keratoconic

cases subjected to the Athens Protocol procedure be-tween 2008 and 2010 were investigated. All proce-dures were performed by the same surgeon (AJK) us-ing the Alcon/WaveLight 400 Hz Eye-Q17 or the EX500 excimer lasers.18 Inclusion criteria were clinical di-agnosis of progressive keratoconus, minimum age of 17 years, and corneal thickness of at least 300 µm. All participants completed an uneventful Athens Protocol procedure and all 231 eyes were observed for up to 3 years. Exclusion criteria were systemic disease, previ-ous eye surgery, chemical injury or delayed epithelial healing, and pregnancy or lactation (female patients).

MEASUREMENTS AND ANALYSISPostoperative evaluation included uncorrected dis-

tance visual acuity (UDVA), manifest refraction, cor-rected distance visual acuity (CDVA) with this refrac-tion, and slit-lamp biomicroscopy for clinical signs of CXL.12 For the quantitative assessment of the induced corneal changes, postoperative evaluation was per-formed by a Pentacam Scheimpflug imaging device (Oculyzer II, WavLight AG, Erlangen, Germany)19 and processed via Examination Software (Version 1.17r47). Specific anterior surface irregularity indices provided by the Scheimpflug imaging analysis were evaluated in addition to keratometric and pachymetric values. These indices are employed in grading and classifica-tion based on the Amsler and Krumeich criteria.20,21 These are the Index of Surface Variance, an expression of anterior surface curvature irregularity, and the In-dex of Height Decentration, calculated with Fourier analysis of corneal height (expressed in microns) to

quantify the degree of cone decentration.22 Index of Surface Variance and Index of Height Decentration were computed for the 8-mm diameter zone.

Descriptive and comparative statistics, analysis of variance, and linear regression were performed by Minitab version 16.2.3 (MiniTab Ltd., Coventry, UK) and Origin Lab version 9 (OriginLab Corp., Northamp-ton, MA). Paired analysis P values less than .05 were considered statistically significant. Visual acuity is re-ported decimally and keratometry in diopters (D). Re-sults are reported as mean ± standard deviation and range as minimum to maximum.

RESULTSThe 231 eyes enrolled belonged to 84 female and 147

male participants. Mean participant age at the time of the operation was 30.1 ± 7.5 years (range: 17 to 57 years). All eyes were followed up to the 3-year follow-up.

VISUAL ACUITY CHANGESTable 1 presents preoperative and postoperative

UDVA and CDVA. UDVA increased by +0.38 ± 0.31 (range: -0.34 to +1.10) and CDVA by +0.20 ± 0.21 (range: -0.32 to +0.90). Figure 1 illustrates, in the form of box plots, visual acuity gained or lost 3 years postopera-tively. These graphs (the 95% median confidence range box) indicate that 95% of the cases had at least +0.1 in-crease in UDVA and 95% had positive change in CDVA.

KERATOMETRIC AND ANTERIOR SURFACE INDICES PROGRESSION

Anterior keratometry continued to flatten over the 3-year follow-up (Figure 2). Descriptive statistics for anterior keratometry, preoperatively and postopera-tively, are presented in Table A (available in the online version of this article).

The values for the Index of Surface Variance and the Index of Height Decentration continued to

TABLE 1Visual Acuity Data (N = 231 Eyes)a

PostoperativeValue Preop 1 Month 3 Months 6 Months 12 Months 24 Months 36 MonthsUDVA Average 0.18 0.42 0.49 0.55 0.57 0.59 0.59 SD (±) ±0.20 ±0.27 ±0.29 ±0.29 ±0.28 ±0.28 ±0.28 Gain/loss n/a +0.23 +0.30 +0.36 +0.36 +0.39 +0.38CDVA Average 0.62 0.69 0.76 0.80 0.81 0.82 0.82 SD (±) ±0.23 ±0.22 ±0.20 ±0.20 ±0.19 ±0.19 ±0.19 Gain/loss n/a +0.07 +0.14 +0.18 +0.18 +0.19 +0.20

preop = preoperative; UDVA = uncorrected distance visual acuity; SD = standard deviation; n/a = not applicable; CDVA = corrected distance visual acuity

aExpressed as the difference of postoperative minus preoperative values (gain/loss). Units are decimal.



decrease over time (Figure 3, Table B, available in the online version of this article).

PACHYMETRIC PROGRESSIONThe thinnest corneal decreased as a result of ex-

cimer laser ablation but then stabilized over time with-out additional thinning (Table 2).

DISCUSSIONMany reports describe the effects of CXL with or

without same-session excimer laser ablation corneal normalization.3 There is general consensus that the in-tervention strengthens the cornea, helps arrest the ec-tasia progression, and improves corneal keratometric values, refraction, and visual acuity.

The key question is the long-term stability of these in-duced changes. For example, is the cornea ‘inactive’ after

the intervention and, if not, is there steepening or flatten-ing and/or thickening or thinning? These issues are even more applicable in the case of the Athens Protocol, due to the partial corneal surface ablation. Ablating a thin, ectatic cornea may sound unorthodox. However, the goal of the topography-guided ablation is to normalize the an-terior cornea and thus help improve visual rehabilitation to a step beyond that a simple CXL would provide.

This study aims to address some of the above issues. The large sample and follow-up time permit sensitive analysis with confident conclusion of postoperative ef-ficacy. We monitored visual acuity changes and for the quantitative assessment we chose to standardize on one Scheimpflug screening device and to focus on key pa-rameters of visual acuity, keratometry, pachymetry, and anterior surface indices.23 All of these parameters reflect changes induced by the procedure and describe postoper-ative progression. However, variations in the two anterior surface indices (Index of Surface Variance and Index of Height Decentration) may provide a more valid analysis than keratometry and visual function.24 Our results indi-cate that the apparent disadvantage of thinning the cornea is balanced by a documented long-term rehabilitating im-provement and synergy from the CXL component.

VISUAL ACUITY CHANGESBased on our results, the Athens Protocol appears

to result in postoperative improvement in both UDVA and CDVA. Average gain/loss in visual acuity was con-sistently positive, starting from the first postoperative month, with gradual and continuous improvement to-ward the 3-year visit. These visual rehabilitation im-provements appear to be superior to those reported in cases of simple CXL treatment.25

However, it is noted that the visual acuity presented with large variations. The standard deviation of UDVA

Figure 1. Change (gain/loss) in visual acuity, expressed as the differ-ence of postoperative minus preoperative values (expressed decimally), showing median level (indicated by (+), average symbol (x), 95% median confidence range box (red borderline boxes), and interquartile intervals range box (black borderline boxes).

Figure 2. Anterior keratometry (K1 flat and K2 steep) as measured by the Scheimpflug device (Oculyzer II, WavLight AG, Erlangen, Germany) preop-eratively up to 3 years postoperatively. All units in keratometric diopters (D).

Figure 3. Anterior surface topometric indices Index of Surface Variance (no units) and Index of Height Decentration (units µm) as measured by the Scheimpflug imaging device (Oculyzer II, WavLight AG, Erlangen, Germany) preoperatively, 1 month postoperatively, and up to 36 months postoperatively.



was ±0.20 preoperatively and ±0.28 postoperatively. Likewise, the standard deviation of CDVA was ±0.23 preoperatively and ±0.20 postoperatively.

We theorize that the reason for visual acuity in kera-toconic cases having such large fluctuations (and often being unexpectedly good) can be attributed to a ‘mul-tifocal’ and ‘soft’ (ie, adaptable) cornea, in addition to advanced neural processing in the individual visual system. However, these ‘advantages’ are essentially ne-gated with CXL treatment, which stiffens the cornea. Over time, possibly due to further topography improve-ment and adaptation to the partially normalized cornea, a noteworthy improvement in visual acuity is observed.

KERATOMETRIC AND ANTERIOR SURFACE INDICES PROGRESSION

After the 1-month visit, keratometric values are re-duced. This progressive potential for long-term flatten-ing has been clinically observed in many cases over at least 10 years. Peer-reviewed reports on this matter have been rare and only recent.26,27

The two anterior surface indices, Index of Surface Variance and Index of Height Decentration, also dem-onstrated postoperative improvement. A smaller value is indication of corneal normalization (lower Index of Surface Variance, less irregular surface, lower In-dex of Height Decentration, cone less steep and more central). These changes are therefore suggestive of corneal topography improvement, in agreement with other smaller sample studies.13 Such changes in Index of Surface Variance and Index of Height Decentration have been reported only recently.28

The initial more ‘drastic’ change of the Index of Height Decentration can be justified by the chief objec-tive of surface normalization, cone centering,6 which is noted even by the first month. The subsequent surface normalization, as also indicated by keratometric flat-tening, suggests further anterior surface improvement.

PACHYMETRIC PROGRESSIONAs expected by the fact that Athens Protocol in-

cludes a partial stromal excimer ablation, there is

reduction of postoperative corneal thickness, mani-fested by the thinnest corneal thickness. What seemed to be a ‘surprising’ result is that the cornea appears to rebound, by gradually thickening, up to 3 years post-operatively. Postoperative corneal thickening after the 1-month ‘lowest thickness baseline’ has also been discussed recently.29,30 In another report,31 the lowest thinnest corneal thickness was noted at the 3-month interval. In that study of 82 eyes treated only with CXL, the average cornea thickened by +24 µm after 1 year compared to the 3-month baseline. In our study of 212 eyes treated with the Athens Protocol procedure, the cornea thickening rate after the baseline first post-operative month was approximately half (+12 µm over the first year), in agreement with a recent publication.29

Therefore, it is possible that stromal changes ini-tiated by the CXL procedure are not just effective in halting ectasia, but are prompting corneal surface flattening and thickening, which appears to be longer lasting than anticipated.

We note, however, that CXL alone results not just in corneal reshaping, but also in stromal density and refrac-tive index, both possibly influencing the reported thick-ness by the Scheimpflug device. Therefore, true corneal thickness differences may not be accurately explored by Scheimpflug imaging due to the principle of operation (densitometry), which has been our clinical experience with abnormal density corneas (ie, corneal scars or ar-cus senilis corneas). Further studies of corneal thickness chances by modalities, such as anterior segment optical coherence tomography, which currently also measure epithelial thickness,32 may be warranted. In addition, cor-neal biomechanical analysis and corneal volume studies may be necessary to further validate such findings.

Our study indicates a significant improvement in all parameters studied. The changes induced by the pro-cedure indicate a consistent trend toward improved visual rehabilitation, corneal flattening (validating ec-tasia arrest), and anterior surface improvement. The Athens Protocol procedure demonstrates impressive refractive, keratometric, and topometric results. Pro-gressive potential for long-term flattening documented

TABLE 2Thinnest Corneal Thickness Measured by the Scheimpflug Device (N = 231 Eyes) (µm)

PostoperativeValue Preoperative 1 Month 3 Months 6 Months 12 Months 24 Months 36 MonthsAverage 451.91 353.95 356.67 364.92 367.15 370.52 370.52SD ±40.02 ±53.90 ±56.41 ±53.64 ±55.70 ±57.84 ±58.21Maximum 547 480 501 501 490 500 500Minimum 297 196 179 220 216 218 218

SD = standard deviation



in this study suggests employment of caution in the surface normalization process to avoid overcorrection.

AUTHOR CONTRIBUTIONSStudy concept and design (AJK, GA); data collection (AJK, GA); analysis and interpretation of data (AJK, GA); drafting of the manuscript (GA); critical revision of the manuscript (AJK, GA); statistical expertise (GA); administrative, technical, or material support (AJK); supervision (AJK)

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and etiology of keratoconus. Int J Keratoco Ectatic Corneal Dis. 2012;1:7-15.

2. Katsoulos C, Karageorgiadis L, Mousafeiropoulos T, Vasileiou N, Asimellis G. Customized hydrogel contact lenses for kera-toconus incorporating correction for vertical coma aberration. Ophthalmic Physiol Opt. 2009;29:321-329.

3. Chan E, Snibson GR. Current status of corneal collagen cross-link-ing for keratoconus: a review. Clin Exp Optom. 2013;96:155-164.

4. Kanellopoulos AJ. Collagen cross-linking in early keratoconus with riboflavin in a femtosecond laser-created pocket: initial clinical results. J Refract Surg. 2009;25:1034-1037.

5. Kanellopoulos AJ, Binder PS. Collagen cross-linking (CCL) with sequential topography-guided PRK: a temporizing alternative for keratoconus to penetrating keratoplasty. Cornea. 2007;26:891-895.

6. Kanellopoulos AJ. Comparison of sequential vs same-day simul-taneous collagen cross-linking and topography-guided PRK for treatment of keratoconus. J Refract Surg. 2009;25:S812-S818.

7. Labiris G, Giarmoukakis A, Sideroudi H, Gkika M, Fanariotis M, Kozobolis V. Impact of keratoconus, cross-linking and cross-linking combined with photorefractive keratectomy on self-re-ported quality of life. Cornea. 2012;31:734-739.

8. Kanellopoulos AJ, Binder PS. Management of corneal ectasia after LASIK with combined, same-day, topography-guided par-tial transepithelial PRK and collagen cross-linking: the athens protocol. J Refract Surg. 2011;27:323-331.

9. Ewald M, Kanellopoulos J. Limited topography-guided surface ablation (TGSA) followed by stabilization with collagen crosslink-ing with UV irradiation and ribofl avin (UVACXL) for keratoconus (KC). Invest Ophthalmol Vis Sci. 2008;49:E-Abstract 4338.

10. Kanellopoulos AJ. Long term results of a prospective randomized bilateral eye comparison trial of higher fluence, shorter duration ultraviolet A radiation, and riboflavin collagen cross linking for progressive keratoconus. Clin Ophthalmol. 2012;6:97-101.

11. Krueger RR, Kanellopoulos AJ. Stability of simultaneous to-pography-guided photorefractive keratectomy and riboflavin/UVA cross-linking for progressive keratoconus: case reports. J Refract Surg. 2010;26:S827-S832.

12. Kanellopoulos AJ, Asimellis G. Introduction of quantitative and qualitative cornea optical coherence tomography findings, in-duced by collagen cross-linking for keratoconus; a novel effect measurement benchmark. Clin Ophthalmol. 2013;7:329-335.

13. Greenstein SA, Fry KL, Hersh PS. Corneal topography indices af-ter corneal collagen crosslinking for keratoconus and corneal ecta-sia: one-year results. J Cataract Refract Surg. 2011;37:1282-1290.

14. Guedj M, Saad A, Audureau E, Gatinel D. Photorefractive kera-tectomy in patients with suspected keratoconus: five-year fol-low-up. J Cataract Refract Surg. 2013;39:66-73.

15. Alessio G, L’abbate M, Sborgia C, La Tegola MG. Photorefrac-tive keratectomy followed by cross-linking versus cross-linking alone for management of progressive keratoconus: two-year follow-up. Am J Ophthalmol. 2013;155:54-65.

16. Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg. 2011;37:149-160.

17. Kanellopoulos AJ. Topography-guided hyperopic and hyper-opic astigmatism femtosecond laser-assisted LASIK: long-term experience with the 400 Hz eye-Q excimer platform. Clin Oph-thalmol. 2012;6:895-901.

18. Kanellopoulos AJ, Asimellis G. Long-term bladeless LASIK outcomes with the FS200 femtosecond and EX500 Excimer Laser workstation: the Refractive Suite. Clin Ophthalmol. 2013;7:261-269.

19. Kanellopoulos AJ, Asimellis G. Correlation between central cor-neal thickness, anterior chamber depth, and corneal keratometry as measured by Oculyzer II and WaveLight OB820 in preopera-tive cataract surgery patients. J Refract Surg. 2012;28:895-900.

20. Krumeich JH, Daniel J, Knülle A. Live-epikeratophakia for kera-toconus. J Cataract Refract Surg. 1998;24:456-463.

21. Faria-Correia F, Ramos IC, Lopes BT, et al. Topometric and to-mographic indices for the diagnosis of keratoconus. Int J Kerat Ectatic Dis. 2012;1:100-106.

22. Kanellopoulos AJ, Asimellis G. Revisiting keratoconus diagno-sis and progression classification based on evaluation of corneal asymmetry indices, derived from Scheimpflug imaging in kera-toconic and suspect cases. Clin Ophthalmol. 2013;7:1539-1548.

23. Markakis GA, Roberts CJ, Harris JW, Lembach RG. Comparison of topographic technologies in anterior surface mapping of ker-atoconus using two display algorithms and six corneal topogra-phy devices. Int J Kerat Ectatic Dis. 2012;1:153-157.

24. Ambrósio R Jr, Caiado AL, Guerra FP, et al. Novel pachymetric parameters based on corneal tomography for diagnosing kerato-conus. J Refract Surg. 2011;27:753-758.

25. Legare ME, Iovieno A, Yeung SN, et al. Corneal collagen cross-linking using riboflavin and ultraviolet A for the treatment of mild to moderate keratoconus: 2-year follow-up. Can J Ophthal-mol. 2013;48:63-68.

26. Vinciguerra P, Albè E, Trazza S, et al. Refractive, topographic, to-mographic, and aberrometric analysis of keratoconic eyes under-going corneal cross-linking. Ophthalmology. 2009;116:369-378.

27. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen cross-linking with riboflavin and ultraviolet-A light in keratoconus: longterm results. J Cataract Refract Surg. 2008;34:796-801.

28. Kanellopoulos AJ, Asimellis G. Comparison of Placido disc and Scheimpflug image-derived topography-guided excimer laser surface normalization combined with higher influence CXL: The Athens Protocol, in progressive keratoconus cases. Clin Ophthalmol. 2013;7:1539-1548.

29. Mencucci R, Paladini I, Virgili G, Giacomelli G, Menchini U. Corneal thickness measurements using time-domain ante-rior segment OCT, ultrasound, and Scheimpflug tomographer pachymetry before and after corneal cross-linking for keratoco-nus. J Refract Surg. 2012;28:562-566.

30. O’Brart DP, Kwong TQ, Patel P, McDonald RJ, O’Brart NA. Long-term follow-up of riboflavin/ultraviolet A (370 nm) cor-neal collagen cross-linking to halt the progression of keratoco-nus. Br J Ophthalmol. 2013;97:433-437.

31. Greenstein SA, Shah VP, Fry KL, Hersh PS. Corneal thickness changes after corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg. 2011;37:691-700.

32. Kanellopoulos AJ, Asimellis G. Anterior segment optical coher-ence tomography: assisted topographic corneal epithelial thick-ness distribution imaging of a keratoconus patient. Case Rep Ophthalmol. 2013;4:74-78.

TABLE AAnterior Keratometry (K) Measured by the Scheimpflug Device (N = 231 Eyes)a

Postoperative

Value Preop 1 Month 3 Months 6 Months 12 Months 24 Months 36 Months

K1 (flat)

Average 46.56 44.44 43.99 43.73 43.54 43.35 43.22

SD ±3.83 ±3.97 ±3.86 ±3.76 ±3.73 ±3.74 ±3.80

Maximum 58.30 55.50 53.75 53.70 53.70 53.70 53.70

Minimum 39.75 36.10 36.10 36.10 36.10 36.10 36.00

K2 (steep)

Average 50.71 47.61 47.03 46.84 46.63 46.44 46.30

SD ±5.14 ±5.15 ±4.99 ±4.92 ±4.91 ±4.88 ±4.91

Maximum 66.62 62.75 61.25 60.25 60.00 60.00 60.00

Minimum 42.60 38.00 37.90 37.90 37.90 37.90 37.20

preop = preoperative; SD = standard deviation aAll units in keratometric diopters (D).

TABLE BAnterior Surface Topometric Indices and Postoperative Progression (N = 231 Eyes)

Postoperative

Value Preoperative 1 Month 3 Months 6 Months 12 Months 24 Months 36 Months

Index of Surface Variance

Average 98.48 83.08 81.08 79.51 78.19 77.22 76.80

SD ±43.47 ±38.73 ±38.13 ±37.79 ±38.04 ±38.28 ±38.41

Maximum 208 190 190 190 190 190 190

Minimum 17 18 17 16 15 14 7

Index of Height Decentration (µm)

Average 0.091 0.063 0.061 0.059 0.058 0.058 0.057

SD ±0.053 ±0.041 ±0.040 ±0.040 ±0.040 ±0.040 ±0.040

Maximum 0.275 0.208 0.208 0.208 0.208 0.208 0.208

Minimum 0.006 0.001 0.001 0.001 0.001 0.001 0.001

SD = standard deviation

© 2013 Kanellopoulos and Asimellis, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

Clinical Ophthalmology 2013:7 1539–1548

Clinical Ophthalmology

Revisiting keratoconus diagnosis and progression classification based on evaluation of corneal asymmetry indices, derived from Scheimpflug imaging in keratoconic and suspect cases

Anastasios John Kanellopoulos1,2

George Asimellis1

1Laservision.gr Eye Institute, Athens, Greece; 2New York University School of Medicine, New York, NY, USA

Correspondence: Anastasios John Kanellopoulos Laservision.gr Institute, 17 Tsocha str, Athens 11521, Greece Tel 30 210 747 2777 Fax 30 210 747 2789 Email [email protected]

Purpose: To survey the standard keratoconus grading scale (Pentacam®-derived Amsler–Krumeich stages) compared to corneal irregularity indices and best spectacle-corrected distance visual acuity (CDVA).Patients and methods: Two-hundred and twelve keratoconus cases were evaluated for keratoconus grading, anterior surface irregularity indices (measured by Pentacam imaging), and subjective refraction (measured by CDVA). The correlations between CDVA, keratometry, and the Scheimpflug keratoconus grading and the seven anterior surface Pentacam-derived topometric indices – index of surface variance, index of vertical asymmetry, keratoconus index, central keratoconus index, index of height asymmetry, index of height decentration, and index of minimum radius of curvature – were analyzed using paired two-tailed t-tests, coefficient of determination (r2), and trendline linearity.Results: The average standard deviation CDVA (expressed decimally) was 0.626 0.244 for all eyes (range 0.10–1.00). The average flat meridian keratometry was (K1) 46.7 5.89 D; the average steep keratometry (K2) was 51.05 6.59 D. The index of surface variance and the index of height decentration had the strongest correlation with topographic keratoconus grading (P 0.001). CDVA and keratometry correlated poorly with keratoconus severity.Conclusion: It is reported here for the first time that the index of surface variance and the index of height decentration may be the most sensitive and specific criteria in the diagnosis, progres-sion, and surgical follow-up of keratoconus. The classification proposed herein may present a novel benchmark in clinical work and future studies.Keywords: diagnosis and classification, Pentacam topometric indices, Amsler–Krumeich keratoconus grading, surface variance, vertical asymmetry, keratoconus index, central keratoconus index, height asymmetry, height decentration, minimum radius of curvature

IntroductionKeratoconus is described as a degenerative bilateral, progressive, noninflammatory corneal disorder characterized by ectasia, thinning, and increased curvature.1,2 It is associated with loss of visual acuity particularly in relation to progressive cornea irregularity,3,4 and usually is manifested asymmetrically between the two eyes of the same patient.5,6 Occasionally, the patient may present with symptoms of photophobia, glare, and monocular diplopia.

The problem of specificity and sensitivity of keratoconus assessment, particularly the diagnosis of early signs of ectasia and/or subclinical keratoconus, and for monitor-ing the progression of the disease, has been extensively studied.7 The commonly used

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options for the clinician include optically-based anterior seg-ment imaging modalities, eg, Placido corneal topography8,9 and slit or Scheimpflug imaging,10 that provide corneal surface qualitative and quantitative data.

Anterior segment topometric indicesRotating camera Scheimpflug imagery (Pentacam, Oculus Optikgeräte GmbH, Wetzlar, Germany), provides a mul-titude of corneal refractive (keratometric), topometric, tomographic, and pachymetric data.11,12 In addition, specific anterior-surface irregularity indices have been developed for the grading and classification of keratoconus development, as well as the post-operative assessment.13–19

The aim of this study was to investigate the values of these indices, the repeatability of their measurement, and their correlation with best spectacle-corrected distance visual acuity (CDVA), keratometry, and commonly used kerato-conus classification in a large pool of clinically diagnosed keratoconic eyes.

Patients and methodsThis study received approval from the Ethics Committee of the authors’ institution, adherent to the tenets of the Declara-tion of Helsinki. Informed consent was obtained from each subject at the time of the first clinical visit. These cases were studied over the span of at least 2 years.

Patient inclusion criteriaThe study group consisted of 212 cases that presented to the authors’ institution. Subjects’ ages ranged from 19–57 years (average 31.9 7.5 years).

Each case was subjected to a complete ocular examina-tion, including subjective refraction, CDVA measurement with this refraction, and slit-lamp biomicroscopy for clinical signs of keratoconus.

Inclusion criteria included a minimum age of 18 years and definite findings consistent with keratoconus, such as those described by the CLECK (Collaborative Longitudinal Evaluation of Keratoconus) group.20 Exclusion criteria included systemic disease, previous corneal surgery, history of chemical injury or delayed epithelial healing, and pregnancy or lactation during the study (for the female patients).

Imaging, measurement, and analysisAnterior segment evaluation, including anterior segment imaging measurements with the Pentacam Scheimpflug rotating camera, was performed on each case. The device was calibrated according to manufacturer’s recommendations

prior to undertaking the measurements. The Pentacam measurements were obtained and processed via the examina-tion software (version 1.17r47).

For each eye, four consecutive measurements were obtained and processed to test for data acquisition repeatability. The default settings, and 25 images per single acquisition were employed.

Linear regression analysis was performed to seek possible correlations. Descriptive and comparative statistics, analysis of variance between keratoconus Amsler–Krumeich stage subgroups, and linear regression were performed with sta-tistics tools provided by Minitab® version 1.6 (Minitab Inc, Coventry, UK) and Origin version 9 (OriginLab Corporation, Northampton, MA, USA). P-values less than 0.05 in the paired analyses were considered statistically significant.

ResultsKeratometric and anterior surface topographic indices statisticsThe sample population consisted of 212 cases, which con-sisted of 65 female and 147 male patients. The preponder-ance towards males in the population is consistent with the authors’ clinical experience in male/female incidence in keratoconic patients21 and keratoconus incidence studies.22 Of the 212 eyes, 113 were the right eye and 99 were the left eye. The average age of all patients was 31.77 7.23 (range 19–57) years.

Table 1 Collective average, standard deviation, maximum, and minimum anterior keratometry and topometric indices, as measured in the 8 mm zone

Average SD Max Min

Anterior cornea K1 – flat (D) 46.78 5.89 78.50 33.70 K2 – steep (D) 51.05 6.59 80.70 42.10 Km – mean (D) 48.80 6.05 78.80 40.60 Astigmatism (D) 2.10 6.05 11.30 12.40Anterior surface topometric indices ISV 98.99 47.43 262 14 IVA (mm) 1.05 0.52 2.52 0.09 KI 1.28 0.17 1.83 0.97 CKI 1.06 0.07 1.30 0.90 IHA (µm) 30.60 22.21 103.00 0.20 IHD (µm) 0.091 0.054 0.256 0.005 Rmin (mm) 6.07 0.88 7.73 3.30CDVA Decimal 0.63 0.25 1.00 0.10

Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; Max, maximum; Min, minimum; Rmin, minimum radius of curvature; SD, standard deviation.


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Table 2 Intraindividual repeatability measurements for the seven anterior surface topometric indices, resulting as the standard deviation (%) of the four consecutive acquisitions on each eye

ISV IVA KI CKI IHA IHD Rmin

Average 2.77% 4.63% 2.15% 2.02% 43.78% 4.67% 1.50%SD ( ) 1.32% 1.94% 1.27% 0.66% 7.18% 1.62% 0.68%

Abbreviations: CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; Rmin, minimum radius of curvature; SD, standard deviation.

Table 3 Coefficient of determination (r2) and Pearson correlation data between best spectacle-corrected distance visual acuity and the seven anterior surface topometric indices within all eyes in the study group (n 212)

CDVA IHD (!m) ISV IVA (mm) KI CKI IHA (!m)

IHDPearson correlation 0.627(r2) 0.393ISVPearson correlation 0.746 0.91(r2) 0.55IVAPearson correlation 0.584 0.893 0.878(r2) 0.340KIPearson correlation 0.680 0.891 0.911 0.845(r2) 0.424CKIPearson correlation 0.642 0.616 0.721 0.436 0.73(r2) 0.396IHAPearson correlation 0.344 0.524 0.484 0.422 0.467 0.252(r2) 0.107RminPearson correlation 0.718 0.799 0.864 0.623 0.790 0.787 0.541(r2) 0.516

Note: P 0.001 in all cases.Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; Rmin, minimum radius of curvature.

The average standard deviation CDVA (expressed decimally) for all eyes was 0.626 0.244 (range 0.10–1.00). Average, standard deviation, maximum and minimum corneal surface keratometry, and topometric indices for all eyes in the study are reported in Table 1. Intraindividual repeatability was assessed via the standard deviation of the four consecutive measurements undertaken for each eye. The average stan-dard deviation of repeatability for the seven topometric indices in all 212 eyes measured is reported in Table 2.

The sample was presented with an average keratometry on the anterior surface flat axis of 46.7 5.89 D and 51.05 6.59 D on the steep axis. The statistical analysis showed that 95% of the sample population had a steep axis keratometry 46.025 D, consistent with the CLEK group standards.20

Topographic indices correlation with CDVAPaired statistics between CDVA and the index of height decentration (IHD), index of surface variance (ISV), index of vertical asymmetry (IVA), keratoconus index (KI), central keratoconus index (CKI), index of height asymmetry (IHA), and minimum radius of curvature (Rmin) (in the 8 mm zone) and specifically the coefficient of determination (r2) and Pearson linear regression data between CDVA and the IHD, ISV, IVA, KI, CKI, IHA, and Rmin indices within all eyes in the study group (n 212) are reported in Table 3.

The correlations between the seven keratoconic anterior surface topometric indices with CDVA are illustrated in Figure 1A–G. Specifically, the scatter and fitted line plots of ISV (Figure 1A), IVA (Figure 1B), KI (Figure 1C), CKI (Figure 1D), IHA (Figure 1E), IHD (Figure 1F), and Rmin (Figure 1G) versus CDVA are plotted, in addition to the 95% confidence (CI) and 95% prediction interval (PI) lines. The paired data present with coefficient of determination (r2) of linear correlation with CDVA of 0.557 for ISV, 0.34 for IVA, 0.424 for KI, 0.396 for CKI, 0.107 for IHA, 0.393 for IHD, and 0.516 for Rmin. The Pearson correlation values versus CDVA were 0.746 for ISV, 0.584 for IVA, 0.680 for KI,

0.642 for CKI, 0.344 for IHA, 0.627 for IHD, and 0.718 for Rmin. In all cases, the P-value was 0.0001 (Table 3).


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Keratoconus classification


2.5

2.0

1.5

1.0

0.5

0.0

IVA

(all

data

)

0.0 0.1 0.2 0.3

IVA = 1.815 − 1.249 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)

Regression95% CI95% PI

0.42648034.0%

33.7%

SR-Sq

R-Sq (adj)

B

1.30

1.25

1.20

1.15

1.10

1.05

1.00

0.95

0.90

CK

I (al

l dat

a)

0.0 0.1 0.2 0.3

CKI = 1.158 − 0.1623 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)


0.049114139.6%39.3%

S

R-Sq

R-Sq (adj)

D

0.30

0.25

0.20

0.15

0.10

0.05

0.00

−0.05

IHD

(pre

oper

ativ

e)

0.0 0.1 0.2 0.3

IHD = 0.1786 − 0.1394 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0CDVA (decimal)

Regression

95% CI95% PI

0.042376939.3%39.0%

SR-SqR-Sq (adj)

F

300

250

200

150

100

ISV

(all

data

)

50

0

0.0 0.1 0.2 0.3

ISV = 189.8 − 145.0 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)

Regression95% CI95% PI31.6518

55.7%55.5%

SR-SqR-Sq (adj)

A

1.8

1.6

1.4

1.2

1.0

0.8

KI (

all d

ata)

0.0 0.1 0.2 0.3

KI = 1.551 − 0.4545 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)


0.12978942.4%42.1%

SR-Sq

R-Sq (adj)

C

125

100

75

50

25

0

IHA

(all

data

)

0.0 0.1 0.2 0.3

IHA = 49.87 − 31.42 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)


22.294710.7%

10.3%

SR-Sq

R-Sq (adj)

E

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

Rm

in (a

ll da

ta) (

mm

)

0.0 0.1 0.2 0.3

Rmin = 4.447 + 2.592 CDVA

0.4 0.5 0.6 0.7 0.8 0.9 1.0

CDVA (decimal)


0.61446051.6%51.3%

SR-SqR-Sq (adj)

G

Figure 1 Scatter and fitted line plot of the seven anterior surface parameters versus CDVA with 95% CI and 95% PI. (A) ISV versus CDVA. (B) IVA versus CDVA. (C) KI versus CDVA. (D) CKI versus CDVA. (E) IHA versus CDVA. (F) IHD versus CDVA. (G) Rmin versus CDVA.Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CI, confidence interval; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; PI, prediction interval; Rmin, minimum radius of curvature.


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Topographic indices correlation with keratoconus stages gradingThe 212 eyes were subjected to keratoconus Amsler– Krumeich stages grading23 by the Pentacam software. The resulting grading designated n1 ten eyes as Stage I, n12 eleven eyes as Stage I–II, n2 32 eyes as Stage II, n23 22 eyes as Stage II–III, n3 54 eyes as Stage III, n34 45 eyes as Stage III–IV, and n4 four eyes as Stage IV.

The correlations between CDVA and the seven Scheimp-flug anterior surface keratometric and topometric indices with the above keratoconus grading are illustrated in Figure 2A–H. Descriptive statistics for the keratoconus grading subgroups for CDVA and the seven topometric indices are presented in Table 4, and two-sample t-test results (not assuming equal variances) between the keratoconus grading subgroups for CDVA and the seven topometric indices are presented in Table 5.

DiscussionThe Pentacam software compares the measured values with the means and standard deviations of a normal population, and helps provide color-coded “flags.” For example, mea-sured values which exceed the standard deviation by a factor of more than 2.5 are classified as abnormal and highlighted in yellow, and pathological values, ie, values that exceed the standard deviation by a factor of more than three, are high-lighted in red. Namely, these indices are the following:

ISV: the unitless standard deviation of individual corneal sagittal radii from the mean curvature. ISV is thus an expression of the corneal surface irregularity. It is elevated in all types of corneal surface irregularity (eg, scars, astig-matism, deformities caused by contact lenses, pachymetry). According to the manufacturer’s user manual,24 an ISV larger than 37 is considered abnormal (marked with yellow) and larger than 41 is pathological (marked with red).IVA: measure (expressed in mm) of the mean difference between superior and inferior corneal curvature (similar to the commonly used inferior/superior ratio).25 IVA is thus the value of curvature symmetry, with respect to the horizontal meridian as the axis of reflection. An IVA larger than 0.28 is considered abnormal, and larger than 0.32 is pathological.KI: a unitless index is expressing the ratio between mean radius values in the upper and lower segment (r sagittal superior to r sagittal inferior). A KI value larger than 1.07 is considered abnormal and/or pathological.CKI: the ratio between mean radius values in a peripheral ring divided by a central ring: r sag (mean peripheral) to r sag mean center (no units). CKI is elevated especially

in central pachymetric, and increases with the severity of central keratoconus. A CKI value larger than 1.03 is considered abnormal and/or pathological.IHA: the mean difference between height values superior minus height values inferior with horizontal meridian as mir-ror axis (expressed in µm)’. IHA is calculated by the height data symmetry comparison of the superior and inferior area, and provides the degree of symmetry of height data with respect to the horizontal meridian as the axis of reflection. IHA is similar to the IVA but based on corneal elevation, and is thus more sensitive. An IHA value larger than 19 is considered abnormal, and larger than 21 is pathological.IHD: the value of the decentration of elevation data in the vertical direction (expressed in µm), and is calculated from a Fourier analysis. This index provides the degree of decentration in the vertical direction, calculated on a ring with radius 3 mm. An IHD value larger than 0.014 is considered abnormal, and larger than 0.016 is pathological.Rmin: expressed in mm. It is a measurement of the small-est radius of sagittal corneal curvature (ie, the maximum steepness of the cone). Values of Rmin less than 6.71 mm are considered abnormal and/or pathological, considering that the average radius of the anterior corneal surface is 7.87 0.27 mm.26

Association with these indices with clinical keratoconus observations is provided by the manufacturer and is listed in Table 6.

The clinical suspicion of early-stage keratoconus may be based on refraction criteria such as a change in refractive power and the axis of astigmatism, fluctuating refraction, and several clinical findings (eg, conspicuous retinoscopy signs). Optical imaging, such as topometry and topography, provides valuable supplementary information, and it has long been supported that the contribution of proper evaluation and analysis of anterior surface irregularity derived from topography,27 or more recently from Pentacam topometry (eg, the seven topographic indices studied in this manuscript), may provide an invaluable aid in the diagnosis and progres-sion evaluation of the disease.28 Only two reports have been identified that address this matter of correlation of the above Pentacam-derived indices with CDVA29 and the severity of keratoconus classification.30

The correlation between the seven anterior surface topo-graphic indices and CDVA appears not very strong in our study. The Rmin (r2 0.516, P 0.001) and ISV (r2 0.557, P 0.001) were found to be the strongest correlated indices with CDVA, in comparison to the other indices. The least


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1.9

1.77

1.48

1.33

1.26

1.171.13

1.1

1.8

1.7

1.6

1.5

1.4

1.3

1.2

1.1

1.0KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

KI d

ata

D

1.35

1.21

1.12

1.081.055

1.021.021.01

1.30

1.25

1.20

1.15

1.10

1.05

1.00

0.95

0.90KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

CK

I dat

a

E1.00.95

0.73 0.7150.675

0.57

0.38

0.22

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

CD

VA

(dec

imal

)

A

Figure 2 Box plots of measured parameters versus keratoconus grading, as produced by the Oculyzer™ software, showing median level (indicated by �), average symbol (�), 95% median confidence range box (black line boxes), and interquartile intervals range box (red line boxes). (A) CDVA versus keratoconus grading. (B) ISV versus keratoconus grading. (C) IVA versus keratoconus grading. (D) KI versus keratoconus grading. (E) CKI versus keratoconus. (F) IHA versus keratoconus grading. (G) IHD versus keratoconus grading. (H) Rmin versus keratoconus grading.Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KC1, keratoconus grading Stage I; KC1–2, keratoconus grading Stage I–II; KC2, keratoconus grading Stage II, KC2–3, keratoconus grading Stage II–III; KC3, keratoconus grading Stage III; KC3–4, keratoconus grading Stage III–IV; KC4, keratoconus grading Stage IV; KI, keratoconus index; PI, prediction interval; Rmin, minimum radius of curvature.

270

4151

74.5

94

12.5

156

205.5

245

220

195

170

145

120

95

70

45

20KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

ISV

dat

a

B

2.5

2.025

1.57

1.25

1.09

0.85

0.59

0.415

2.0

1.5

1.0

0.5

0.0KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

IVA

dat

a

C

100

57.85

36.7

29.6

37.5

25.15

1718.5

90

80

70

60

50

40

30

20

10

0

KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

IHA

dat

a

F

0.250

0.03350.045

0.0665

0.225

0.200

0.175

0.150

0.125

0.100

0.075

0.025

0.050

0.000KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

IHD

dat

a

G

0.09150.1055

0.147

0.245

8.0

6.9456.71

6.48

6.095.835

5.32

4.38

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0KC1 KC1–2 KC2 KC2–3 KC3 KC3–4 KC4

Rm

in (m

m)

H


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Table 4 Descriptive statistics for the keratoconus grading subgroups for age, best spectacle-corrected distance visual acuity, and the seven anterior surface topometric indices

Keratoconus grading

Stage I Stage I–II Stage II Stage II–III Stage III Stage III–IV Stage IV

Age (years) Mean 31.0 32.5 31.3 31.3 30.9 30.6 31.0 SD ( ) 8.4 7.6 7.8 6.0 6.7 7.3 8.4CDVA Mean 0.907 0.779 0.695 0.64 0.55 0.416 0.2225 SD ( ) 0.107 0.166 0.202 0.133 0.188 0.187 0.0634 SEM 0.034 0.05 0.036 0.028 0.026 0.028 0.032ISV Mean 40.6 50.91 72.75 93.77 114.07 157.8 218.8 SD ( ) 2.88 3.56 9.32 5.25 9.51 17.9 29 SEM 0.91 1.1 1.6 1.1 1.3 2.7 14IVA Mean 0.422 0.5618 0.855 1.068 1.219 1.612 2.002 SD ( ) 0.0735 0.0994 0.134 0.194 0.258 0.361 0.497 SEM 0.023 0.03 0.024 0.041 0.035 0.054 0.25KI Mean 1.10 1.12 1.1838 1.2664 1.3291 1.47 1.7475 SD ( ) 0.0163 0.0335 0.0522 0.027 0.0547 0.121 0.0929 SEM 0.0052 0.01 0.0092 0.0058 0.0075 0.018 0.046CKI Mean 1.014 1.0145 1.0291 1.05 1.0711 1.1269 1.208 SD ( ) 0.0135 0.023 0.0352 0.0385 0.0394 0.0702 0.101 SEM 0.0043 0.0069 0.0062 0.0082 0.0054 0.01 0.051IHA Mean 18.05 20.55 26.5 33.3 37.7 39.3 61.7 SD ( ) 9.66 7.68 15.6 17.3 19.3 28.8 14.4 SEM 3.1 2.3 2.7 3.7 2.6 4.3 7.2IHD Mean 0.031 0.04355 0.0677 0.0878 0.1063 0.1512 0.24 SD ( ) 0.00723 0.00972 0.0183 0.0169 0.0266 0.0373 0.0185 SEM 0.0023 0.0029 0.0032 0.0036 0.0036 0.0056 0.0093Rmin (mm) Mean 6.922 6.755 6.52 6.104 5.775 5.146 4.165 SD ( ) 0.311 0.275 0.463 0.483 0.381 0.675 0.513 SEM 0.098 0.083 0.082 0.1 0.052 0.1 0.26

Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; Rmin, minimum radius of curvature; SD, standard deviation; SEM, standard error of the mean.

correlated index was IHA (r2 0.107, P 0.001). This could be a result of significantly worse intraindividual repeatability of this index, possibly as a result of the complicated nature of its algorithm. As indicated in Table 2, repeatability, measured by the standard deviation of the measured index over four consecutive acquisitions, was as good as 1.50% on average for Rmin and up to 4.6% for IHD and IVA; however, the IHA index had a distinctive 43.78% average standard deviation among any four consecutive measurements, indicating very poor repeatability. In addition, the IHA index had the worst correlation with keratoconus severity (Figure 2F) with two stages not statistically significant and the remaining also borderline.

This study indicates that the correlation between keratoconus severity, and possibly very early progression indicators, and the anterior surface topographic indices can be better described with the ISV (with the exception of the highest stage [Stage IV], all other P-values were 0.001), followed by the IHD (with the exception of the lowest stage [Stage I], all other P-values were 0.001).

In addition, our results indicate that the visual perfor-mance in keratoconus was not clearly predicable for kera-toconus severity, and thus CDVA cannot be a dependable indicator of keratoconus severity and/or progression and therefore cannot be employed in postoperative assessment aiming to arrest keratoconus progression by, for example,


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Table 5 Two-sample t-test results, not assuming equal variances, between successive pairs of keratoconus severity stages subgroups for best spectacle-corrected distance visual acuity and the seven anterior surface topometric indices

Stage I vs I–II Stage I–II vs II Stage II vs II–III Stage II–III vs III Stage III vs III–IV Stage III–IV vs IV

CDVAEstimate for difference 0.1279 0.0838 0.0558 0.0892 0.1344 0.193595% CI for difference ( 0.2552, 0.0006) ( 0.2115, 0.0440) ( 0.1473, 0.0357) ( 0.1657, 0.0127) ( 0.2095, 0.0593) ( 0.2890, 0.0980)t-value 2.12 1.36 1.22 2.34 3.55 4.58P-value 0.049 0.187 0.227 0.023 0.001 0.001ISVEstimate for difference 10.31 21.84 21.02 20.3 43.7 6195% CI for difference (7.35, 13.27) (17.87, 25.82) (17.02, 25.03) (16.88, 23.72) (37.79, 49.62) (14.1, 107.8)t-value 7.33 11.1 10.55 11.86 14.76 4.14P-value 0.001 0.001 0.001 0.001 0.001 0.026IVAEstimate for difference 0.1398 0.2929 0.2135 0.1503 0.3939 0.3995% CI for difference (0.0602, 0.2195) (0.2139, 0.3718) (0.1167, 0.3103) (0.0414, 0.2592) (0.2660, 0.5218) ( 0.419, 1.199)t-value 3.69 7.67 4.48 2.77 6.13 1.53P-value 0.002 0.001 0.001 0.008 0.001 0.222KIEstimate for difference 0.02 0.0638 0.0826 0.06271 0.1414 0.277195% CI for difference ( 0.0043, 0.0443) (0.0357, 0.0918) (0.0607, 0.1045) (0.04394, 0.08148) (0.1024, 0.1804) (0.1186, 0.4356)t-value 1.76 4.66 7.59 6.66 7.26 5.56P-value 0.099 0.001 0.001 0.001 0.001 0.011CKIEstimate for difference 0.00055 0.01452 0.0209 0.02111 0.0558 0.080695% CI for difference ( 0.01670, 0.01779) ( 0.00459, 0.03362) (0.0001, 0.0417) (0.00126, 0.04096) (0.0323, 0.0792) ( 0.0842, 0.2454)t-value 0.07 1.56 2.03 2.15 4.75 1.56P-value 0.947 0.131 0.049 0.038 0.001 0.217IHAEstimate for difference 2.5 5.93 6.82 4.38 1.58 22.4495% CI for difference ( 5.58, 10.59) ( 1.37, 13.22) ( 2.45, 16.10) ( 4.74, 13.50) ( 8.45, 11.60) (0.90, 43.98)t-value 0.65 1.65 1.48 0.97 0.31 2.68P-value 0.522 0.108 0.145 0.338 0.755 0.044IHDEstimate for difference 0.01255 0.02411 0.02012 0.01856 0.04487 0.088895% CI for difference (0.00474, 0.02035) (0.01522, 0.03300) (0.01038, 0.02985) (0.00836, 0.02876) (0.03165, 0.05808) (0.0610, 0.1166)t-value 3.38 5.52 4.16 3.64 6.76 8.21P-value 0.003 0.001 0.001 0.001 0.001 0.001Rmin (mm)Estimate for difference 0.167 0.235 0.416 0.329 0.629 0.98195% CI for difference ( 0.438, 0.103) ( 0.473, 0.003) ( 0.681, 0.151) ( 0.563, 0.094) ( 0.855, 0.403) ( 1.858, 0.104)t-value 1.30 2.02 3.16 2.85 5.56 3.56P-value 0.209 0.053 0.003 0.008 0.001 0.038

Abbreviations: CDVA, best spectacle-corrected distance visual acuity; CI, confidence interval; CKI, central keratoconus index; IHA, index of height asymmetry; IHD, index of height decentration; ISV, index of surface variance; IVA, index of vertical asymmetry; KI, keratoconus index; Rmin, minimum radius of curvature; vs, versus.

crosslinking with riboflavin.31 It is customary to assess sever-ity based on visual function and, as noted above, appears to be deceiving. Clinicians must be cautious. For example, the data presented in this work (Figure 2A) suggest that CDVA is not very well correlated with the keratoconus severity, as the spread of CDVA measurements within the same “sever-ity stage” (eg, Stage III, Stage III–IV) was found to be too large, and interfering with neighboring stages. The average coefficient of determination (r2) was in the order of 0.5, in agreement with previous studies.32

The above results are in agreement with the authors’ past clinical experience with a significant number of keratoconic patients followed for over 15 years. The observation has been that visual acuity can present with large variations, and can sometimes be unexpectedly good for the corresponding keratometry and overall corneal asymmetry. This may be due to a “multifocal” and “soft,” (ie, adaptable) cornea in addition to possible advanced neural processing develop-ment in the individual. However, these “advantages” are, to a large degree, compromised by the procedure of crosslinking


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with riboflavin applied in young (18–25 years old) kerato-conus patients in long-term clinical observations.

Although not specifically studied herein, clinical assess-ment of this group is that CDVA is poorly correlated to keratoconus severity mainly in younger patients. As older keratoconus patients were encountered (ie, those over 30 years old and in most of those over 40 years old), their CDVA started to correlate with the ISV and IHD. This observation poses an even greater reason for clinicians to employ the strongest clinical suspicion in younger patients that may be at risk for developing keratoconus and may be labeled “normal” if a very good CDVA performance is maintained.

This may be explained by the higher elasticity of the pathologic cornea in younger keratoconus patients. This age-related difference may allow them to achieve high CDVA values with monocular testing by squinting and/or head tilt-ing. ISV and IHD assessment in younger patients with relative low keratometric values may be the crucial factor in early diagnosis of keratoconus. We have encountered this clinical finding exaggerated in very young (18–22 years) keratoconic patients that had in the past undergone just cross-linking sta-bilization. We theorize that by increasing the biomechanical stability with CXL, these patients start to more appreciate the existing corneal irregularity.31,33,34

ConclusionThe ISV and IHA, both derived from Scheimpflug corneal imaging, may be more sensitive and specific tools than CDVA in evaluating early diagnosis and possible progression in kera-toconus patients and corneal ectasia. They may become a novel benchmark for future studies, and may aid in the development of new keratoconus diagnostic and follow-up criteria.

DisclosureBoth AJK and GA occasionally consult for Alcon/Wave-light. The authors report no other conflicts of interest in this work.

References 1. Krachmer JH, Feder RS, Belin MW. Keratoconus and related

noninflammatory corneal thinning disorders. Surv Ophthalmol. 1984;28(4):293–322.

2. Belin MW, Asota IM, Ambrosio R Jr, Khachikian SS. What’s in a name: keratoconus, pellucid marginal degeneration, and related thinning disorders. Am J Ophthalmol. 2011;152(2):157–162.

3. Ambrosio R Jr, Caldas DL, da Silva RS, Pimentel LN, de Freitas VB. Impacto da análise do “wavefront” na refratometria de pacientes com ceratocone [Impact of the wavefront analysis in refraction of keratoco-nus patients]. Rev Bras Oftalmol. Rio de Janeiro: Sociedade Brasileira de Oftalmologia; 2010;69(5):294–300. Portuguese [with English abstract].

4. Kosaki R, Maeda N, Bessho K, et al. Magnitude and orientation of Zernike terms in patients with keratoconus. Invest Ophthalmol Vis Sci. 2007;48(7):3062–3068.

Table 6 Classification stages of keratoconus adapted from the classical Amsler–Krumeich standards30

CDVA ISV KI Other indices Rmin, mm Retinoscopy signs Cornea slit-lamp observations

Pre-stage (early signs)

20/20– 20/15

30 1.04–1.07 All four indices are “normal”

7.8–6.7 No clear light or shadow movement.Hint of “scissors” effect

Clear cornea, unobtrusive. Horizontal, oval, or round shades central or slightly decentered, when observed under direct ophthalmoscopy

Level 1 20/25– 20/15

30–55 1.07–1.15 Sometimes one value within the “abnormal” range

7.5–6.5 Distorted retinoscopic reflex. Scissors effect

Clear cornea. Fleischer’s ring at the apex base. Cone and cone base clearly visible with direct ophthalmoscopy. Decrease in apex thickness not visible, but measurable

Level 2 20/60– 20/20

55–90 1.10–1.25 Often one value within the “abnormal” range

6.9–5.3 Clear scissors effect, retinoscopy difficult to perform

Often cornea still clear, apex slightly thinner and eventually decentered. Partial or circular Fleischer’s ring. Vogt’s striae may be visible

Level 3 20/125– 20/30

90–150 1.15–1.45 At least one value within the “abnormal” range

6.6–4.8 Distinct scissors effect, retinoscopy nearly impossible to perform

Apex thinner, decentered, and often slightly cloudy. Clear and mostly circular Fleischer’s ring. Vogt’s striae clearly visible. Eventually Munson’s sign may appear

Level 4 20/400– 20/100

150 1.50 At least one value within the “abnormal” range

5.00 Retinoscopy impossible to perform

Cornea often scarred and opaque in the apex. Munson’s sign evident

Notes: Best spectacle-corrected distance visual acuity correction, with topography-based graduation indices (index of surface variance; keratometry index) compared with retinoscopy signs and clinical corneal slit-lamp observations. Adapted from the WaveLight® Allegro Oculyzer™ User Manual.24

Abbreviations: CDVA, best spectacle-corrected distance visual acuity; ISV, index of surface variance; KI, keratoconus index; Rmin, minimum radius of curvature.


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5. Zadnik K, Steger-May K, Fink BA, et al; CLEK Study Group. Between-eye asymmetry in keratoconus. Cornea. 2002;21(7): 671–679.

6. Jones-Jordan LA, Walline JJ, Sinnott LT, Kymes SM, Zadnik K. Asymmetry in keratoconus and vision-related quality of life. Cornea. 2013;32(3):267–272.

7. Randleman JB, Trattler WB, Stulting RD. Validation of the Ectasia Risk Score System for preoperative laser in situ keratomileusis screening. Am J Ophthalmol. 2008;145(5):813–818.

8. Rabinowitz YS. Videokeratographic indices to aid in screening for keratoconus. J Refract Surg. 1995;11(5):371–379.

9. Abad JC, Rubinfeld RS, Del Valle M, Belin MW, Kurstin JM. Vertical D: a novel topographic pattern in some keratoconus suspects. Ophthalmology. 2007;114(5):1020–1026.

10. Ambrosio R Jr, Simonato RS, Luz A, Coca Velarde LG. Corneal-thickness spatial profile and corneal-volume distribution: tomographic indices to detect keratoconus. J Cataract Refract Surg. 2006;32(11): 1851–1859.

11. McAlinden C, Khadka J, Pesudovs K. A comprehensive evaluation of the precision (repeatability and reproducibility) of the Oculus Pentacam HR. Invest Ophthalmol Vis Sci. 2011;52(10):7731–7737.

12. Swartz T, Marten L, Wang M. Measuring the cornea: the latest devel-opments in corneal topography. Curr Opin Ophthalmol. 2007;18(4): 325–333.

13. Li X, Yang H, Rabinowitz YS. Keratoconus: classification scheme based on videokeratography and clinical signs. J Cataract Refract Surg. 2009;35(9):1597–1603.

14. Greenstein SA, Fry KL, Hersh PS. Corneal topography indices after corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg. 2011;37(7):1282–1290.

15. Sonmez B, Doan MP, Hamilton DR. Identification of scanning slit-beam topographic parameters important in distinguishing normal from keratoconic corneal morphologic features. Am J Ophthalmol. 2007;143(3):401–408.

16. Markakis GA, Roberts CJ, Harris JW Jr, Lembach RG. Comparison of topographic technologies in anterior surface mapping of kera-toconus using two display algorithms and six corneal popography devices. International Journal of Keratoconus Ectatic Diseases. 2012;1(3):153–157.

17. Faria-Correia F, Ramos IC, Lopes BT, et al. Topometric and tomo-graphic indices for the diagnosis of keratoconus. International Journal of Keratoconus Ectatic Diseases. 2012;1(2):92–99.

18. Rufer F, Schroder A, Arvani MK, Erb C. Zentrale und periphere hornhautpachymetrie – normevaluation mit dem Pentacam-System [Central and peripheral corneal pachymetry – standard evaluation with the Pentacam system]. Klin Monbl Augenheilkd. Stuttgart: Thieme; 2005;222(2):117–122. German [with English abstract].

19. Emre S, Doganay S, Yologlu S. Evaluation of anterior segment parameters in keratoconic eyes measured with the Pentacam system. J Cataract Refract Surg. 2007;33(10):1708–1712.

20. Zadnik K, Barr JT, Edrington TB, et al. Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) study. Invest Ophthalmol Vis Sci. 1998;39(13):2537–2546.

21. Kanellopoulos AJ, Asimellis G. Introduction of quantitative and qualita-tive cornea optical coherence tomography findings induced by collagen cross-linking for keratoconus: a novel effect measurement benchmark. Clin Ophthalmol. 2013;7:329–335.

22. Gordon-Shaag A, Millodot M, Shneor E. The epidemiology and etiology of keratoconus. International Journal of Keratoconus Ectatic Diseases. 2012;1(1):7–15.

23. Krumeich JH, Daniel J, Knulle A. Live-epikeratophakia for keratoconus. J Cataract Refract Surg. 1998;24(4):456–463.

24. WaveLight GmbH. WaveLight® Allegro Oculyzer™ 1074 User Manual (English). Erlangen: WaveLight GmbH; 2001.

25. Jafri B, Li X, Yang H, Rabinowitz YS. Higher order wavefront aber-rations and topography in early and suspected keratoconus. J Refract Surg. 2007;23(8):774–781.

26. Dubbelman M, Weeber HA, van der Heijde RG, Volker-Dieben HJ. Radius and asphericity of the posterior corneal surface determined by corrected Scheimpflug photography. Acta Ophthalmol Scand. 2002;80(4):379–383.

27. Maeda N, Klyce SD, Smolek MK, Thompson HW. Automated kerato-conus screening with corneal topography analysis. Invest Ophthalmol Vis Sci. 1994;35(6):2749–2757.

28. Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the keratometric index: evaluation by the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg. 2008;34(1):137–145.

29. Lopes BT, Ramos IC, Faria-Correia F, et al. Correlation of topometric and tomographic indices with visual acuity in patients with keratoconus. International Journal of Keratoconus Ectatic Diseases. 2012;1(3): 167–172.

30. Ishii R, Kamiya K, Igarashi A, Shimizu K, Utsumi Y, Kumanomido T. Correlation of corneal elevation with severity of keratoconus by means of anterior and posterior topographic analysis. Cornea. 2012;31(3): 253–258.

31. Kanellopoulos AJ. Comparison of sequential vs same-day simultaneous collagen cross-linking and topography-guided PRK for treatment of keratoconus. J Refract Surg. 2009;25(9):S812–S818.

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34. Kanellopoulos AJ. Post-LASIK ectasia. Ophthalmology . 2007;114:1230.


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Clinical Ophthalmology 2013:7 329–335


Introduction of quantitative and qualitative cornea optical coherence tomography findings induced by collagen cross-linking for keratoconus: a novel effect measurement benchmark

A John Kanellopoulos1,2

George Asimellis1

1Laservision.gr Institute, Athens, Greece; 2New York University Medical School, New York, NY, USA

Correspondence: A John Kanellopoulos Laservision.gr Eye Institute, 17 Tsocha Street, Athens 11521, Greece Tel 30 210 747 2777 Fax 30 210 747 2789 Email [email protected]

Purpose: To introduce a novel, noninvasive technique to determine the depth and extent of anterior corneal stroma changes induced by collagen cross-linking (CXL) using quantitative analysis of high-resolution anterior-segment optical coherence tomography (OCT) post-operative images.Setting: Private clinical ophthalmology practice.Patients and methods: Two groups of corneal cross-sectional images obtained with the OptoVue RTVue anterior-segment OCT system were studied: group A (control) consisted of unoperated, healthy corneas, with the exception of possible refractive errors. The second group consisted of keratoconic corneas with CXL that were previously operated on. The two groups were investigated for possible quantitative evidence of changes induced by the CXL, and specifically, the depth, horizontal extent, as well as the cross-sectional area of intrastromal hyper-reflective areas (defined in our study as the area consisting of pixels with luminosity greater than the mean 2 standard deviation of the entire stromal cross section) within the corneal stroma.Results: In all images of the second group (keratoconus patients treated with CXL) there was evidence of intrastromal hyper-reflective areas. The hyper-reflective areas ranged from 0.2% to 8.8% of the cross-sectional area (mean standard deviation; 3.46% 1.92%). The extent of the horizontal hyper-reflective area ranged from 4.42% to 99.2% (56.2% 23.35%) of the cornea image, while the axial extent (the vertical extent in the image) ranged from 40.00% to 86.67% (70.98% 7.85%). There was significant statistical difference (P 0.02) in these values compared to the control group, in which, by application of the same criteria, the same hyper-reflective area (owing to signal noise) ranged from 0.00% to 2.51% (0.74% 0.63%).Conclusion: Herein, we introduce a novel, noninvasive, quantitative technique utilizing anterior segment OCT images to quantitatively assess the depth and cross-sectional area of CXL in the corneal stroma based on digital image analysis. Mean cross-sectional area showing evidence of CXL was 3.46% 1.92% of a 6 mm long segment.Keywords: Collagen cross-linking, keratoconus, optical coherence tomography, higher fluence cross-linking, cornea ectasia, Athens Protocol

IntroductionKeratoconus (KCN) is a degenerative bilateral, progressive, noninflammatory disorder characterized by ectasia, thinning, and increased curvature of the cornea, and is associated with loss of visual acuity, particularly in relation to high-order aberrations.1–4

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Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A irradiation is a common technique for tissue stabilization.5,6 Several studies have shown that CXL is an effective intervention to halt the progression of keratoconus and corneal ectasia.7

Anterior-segment optical coherence tomography (AS-OCT) is a promising imaging mode providing high-resolution cross-sectional images across a meridian of choices that can be employed in KCN diagnosis.8,9 The most advanced AS-OCT systems invariably employ Fourier spectral-domain signal processing. As of today, there are a number of different spectral domain OCT systems commercially available.10,11

The ability to provide real-time cross sectional mapping, in conjunction with the very principle of operation, namely photon back scattering, provides the understudied application of quantitative assessment of the extent of stromal changes due to CXL.

OCT and CXL demarcation line observationsTo date, the efficacy of CXL treatment can be monitored only indirectly by postoperative follow-up observations, such as with a Scheimpflug camera,12 or with corneal confocal microscopy.13

In addition, a corneal stromal demarcation line indicating the transition zone between cross-linked anterior corneal stroma and untreated posterior corneal stroma can be detected in slit-lamp examination as early as 2 weeks after treatment.14 In our clinical assessment, the presence of this finding over the anterior two-thirds of the stroma confirms that sufficient CXL treatment has occurred.

Following our presentation and the introduction in the peer-reviewed literature of the use of OCT imaging in order to evaluate the CXL-induced demarcation line, OCT has seen some recent interest as a tool for investigating CXL effects, such as corneal thickness before and after CXL for KCN, and demarcation line depth following CXL.15–22

The principle lies in the fact that although these lines do not appear to affect vision, as they correspond to changes in stromal density, they appear as brighter (hyper-reflective) areas on cross-sectional corneal OCT scans. However, the depth and extent of stromal changes induced by CXL has been difficult to evaluate quantitatively in the clinic.

The motivation for our study was to advance this aforementioned theory by examining not only the demarcation line depth between the suspected CXL and the deeper cornea with corneal OCT, but also to attempt to quantitatively

assess the extent of this area on a large number of patients over a large postoperation interval. Our novel technique is based on digital signal processing on cross-sectional OCT images of corneas, and evaluates quantitatively and, in our opinion, free of examiner bias, the extent of CXL changes in the corneal stroma.

MethodsThis prospective interventional case series study received approval by the Ethics Committee of our Institution and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from each subject at the time of the CXL intervention or at the first clinical visit. The study was conducted in our clinical practice on patients during their regular clinical visits (control group) and scheduled postoperative procedure visits (KCN group).

Patient inclusion criteriaThe control group (50 patients, 100 eyes) consisted of patients with eyes with unoperated corneas (ie, normal eyes with no ocular pathology other than refractive error). Mean patient age was 35.2 9.1 years (range 19–48), equally divided between males and females. Before OCT corneal mapping, a complete ocular examination and tomographic topography was performed to screen for corneal abnormalities.

The second group (47 patients, 94 eyes) consisted of KCN patients previously operated with CXL by employing the Athens Protocol, which combined same-day phototherapeutic keratectomy epithelial removal and partial topographically-guided photorefractive keratectomy normalization of the cornea ectasia, followed by high-fluence, short-duration riboflavin induced CXL.21

The mean patient age in this group was 28.1 7.1 years (range 16–45 years). There is a bias towards males in this group (33 males, 14 females), which is consistent with our clinical experience of the male–female incidence of keratoconic


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Figure 1 (A) Typical cornea cross-sectional meridian image of a patient with KCN. (B) The selected hyper-reflective intrastromal area is indicated in red.


patients, and with previous reports.2 Of the 94 eyes included in the study, 47 were oculus sinister and 47 were oculus dexter. Mean postoperative time since CXL operation was 17.65 20.83 months, with a range of 1 to 72 months.

Most patients enrolled in the group had bilateral CXL operation, and thus both eyes were included in the study, while in some patients only one eye was included in the study. For some patients, images from more than one visit were included in the study (separated by at least 3 months).

MaterialsThe OptoVue RTVue (OptoVue Inc, Fremont, CA, USA) AS-OCT system was employed in the study. Using the L-Cam lens, a 6 mm long Hi-Res Cross Line Scan, centered at the pupil center along the vertical meridian, was recorded. The meridional cross-sectional images

were processed with the RTVue software (version 5.1.0, processing algorithm A5, 1, 0, 90). The software averages up to 32 successive acquisitions. In our study, we included images consisting of at least five averages.

Our novel investigation techniqueAll images from both groups were investigated for possible quantitative evidence of changes induced by CXL. Evidence of such was considered as the existence of the intrastromal hyper-reflective demarcation line. To search for such a line, images were loaded into commercially available software, Adobe Photoshop CS5 Version 12.04 (Adobe Systems Inc, San Jose, CA, USA).

For every meridional cross-sectional image, the pixels associated with the stromal cross-section were selected with the marquee tool. The area separated by 10 pixels from the anterior and the exterior corneal surfaces were deselected, as they are typically of higher luminosity (Figure 1). The extent (pixel count) of the selected stromal image area was determined with the histogram tool report. The dialog box for this tool also provides the mean standard deviation of the luminosity for the selected area.

The hyper-reflective demarcation area was quantitatively defined in this study as the population of pixels (pixel count) having luminosity greater than the value defines as luminosity mean 2 standard deviations, as obtained in the previous step.

Table 1 Hyper-reflective area corresponding to meridional corneal cross section, as measured in the two groups

Group A (control) Group B (KCN)

Hyper-reflective area (pixels)

Cross- sectional area (%)

Hyper- reflective area (pixels)

Cross- sectional area (%)

Mean 427 0.74% 2018 3.46%Max 1518 2.50% 4927 8.80%Min 0 0.00% 121 0.18%Stdev 374 0.63% 1105 1.92%

Abbreviations: KCN, keratoconus; Max, maximum; Min, minimum; Stdev, standard deviation.


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10%

9%

8%

7%

6%

5%

4%

3%

2%

1%

0%0 10 20 30

Time of post-operative examination (months)

CX

L ar

ea (%

)

40 50 60 70 80

Figure 2 Demarcation line area as a function of time elapsed since the CXL operation.Note: The trend line shows that over time, this area diminishes.Abbreviation: CXL, cross-linking.


Subsequently, after using the histogram tool report again, the extent (pixel count) of the hyper-reflective intrastromal area was recorded, as well as its horizontal extent (pixels across the y axis), and this was compared to the horizontal extent of the captured image, which was set to a standard of 860 pixels across the y-axis.

Similarly, the axial extent (depth of demarcation line) was assessed and compared to the depth of the corneal section (vertical line in the image; that is, pixels in the x-axis) to which it corresponded.

Descriptive statistics (average, minimum, maximum, standard deviation, bias, and range), comparative statistics, and linear regression were performed in Microsoft Excel 2010 (Microsoft Corp, Redmond, WA, USA) and OriginLab version 8 (OriginLab Corp, Northampton, MA, USA). Analysis of variance between groups was performed using the OriginLab statistics tool.

ResultsAreal extent (depth and diameter) of demarcationCross-sectional meridian area measurements had an average of 59,183 pixels ( 5778), ranging from 80,729 to 39,951 (maximum to minimum). This corresponds to an area of 2.88 mm2. Mean luminosity values were, on a grayscale of 0 to 255, 63 13, ranging from 89 to 25 (maximum to minimum).

As shown in Table 1, the intrastromal hyper-reflective area found with this technique for group A (control) had a mean area of 427.25 373.81 pixels (range, maximum to minimum, 1518-0), corresponding to a mean of 0.74% 0.63% (range, 2.50%–0.00%), corresponding to 0.02 mm2. This contrasts with group B (KCN), in which the mean hyper-reflective area had a mean of 2018.21 1104.70 (range, 4927–121), corresponding to a mean of 3.46% 1.92% (range, 8.80%–0.18%), or 0.098 mm2 of the corneal cross-sectional area. Of the 94 cases examined, 72 had more than 2.50% hyper-reflective areas, whereas six were close to and 16 were below this mark. The two groups were found to be statistically different (comparison of hyper-reflective area pixel count; P 0.02). The over time development of the extent of the area of demarcation; that is, the CXL area over post-operative time, is plotted in Figure 2.

Horizontal extent of demarcationThe horizontal extent of demarcation was assessed for the KCN group and was compared to the standard 860 pixel

Table 2 Horizontal and axial extent of hyper-reflective area in group B (KCN)

CXL width (pixels)

Horizontal extent

V line total

Axial extent % V

Overall width V line CXL

Mean 483.35 56.20% 61.93 43.81 70.98%Max 853 99.19% 80 65 86.67%Min 38 4.42% 38 30 40.00%Stdev 200.82 23.35% 8.18 6.96 7.85%

Abbreviations: KCN, keratoconus; CXL, cross-linking; V, vertical; Max, maximum; Min, minimum; Stdev, standard deviation.

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%0 10 20 30

Time of post-operative examination (months)

Dem

arca

tion

line

dept

h (%

)

40 50 60 70 80

Figure 3 Demarcation line depth as a function of time elapsed since the CXL operation.Note: The trend line shows that over time this depth remains constant to about two-thirds of the corneal depth.Abbreviation: CXL, cross-linking.


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width extent of the corneal cross-section. As shown in Table 2, on average the extent of the hyper-reflective area (CXL width) was 483.35 200.82 pixels (range, 853–38), corresponding to an average of 56.20% of the cross-section width, ranging from 99.19% to 4.42%. Of the subgroup of 78 corneas with more than a 2.50% hyper-reflective area, the minimum was 12.45%.

Axial extent (depth) of demarcationThe axial extent of demarcation corresponds to what we describe as the depth of the CXL effect. The quantitative assessment is subject to the corneal thickness, which varies significantly among images. In each image studied, the corneal thickness was measured in pixels (vertical line total in Table 2), and was found to correspond to an average of 61.93 8.18 pixels (max to min, 80–38). Considering that 6 mm across the image corresponded to 860 pixels, the 61.93 pixels corneal thickness translates to 432 m of thickness.

Having measured the corneal thickness of each individual section, the distance in pixels (vertical line CXL) from the anterior corneal surface was measured. On average, it was found to be 43.81 6.96 pixels (range, 65–30), corresponding to 305.6 m or 70.98% of the total corneal thickness. The over time (postoperative) development of the depth of the area of demarcation, that is CXL area over time, is presented in Figure 3.

DiscussionBy examining high-resolution corneal OCT images, we encountered statistically different findings between the treated group (KCN; group B) and the control group (A).

It appears that there is a statistically signif icant difference between the control group and the KCN group regarding the presence of a demarcation line, as quantitatively measured by the extent of the area of the hyper-reflective demarcation line, indicating a localized

change in stromal (treated) density over the underlying (untreated) stroma.

In 72 of 94 cases, the demarcation line area corresponded to more than 2.50% of the total corneal cross-sectional area, with a mean standard deviation of 3.46% 1.92%. In the entire control group A, by applying the same luminosity criteria, the similar area had a mean of 0.74% 0.63%. We believe that these pixel counts represent merely signal noise rather than reflect actual changes in stromal density. Thus, we can ascertain that the demarcation line viewed by OCT can be a good indication of the extent of collagen density changes induced by CXL.

Over time, these density changes become less apparent. The trend line shown in Figure 2 has a negative slope (reduced

Figure 4 Example of epithelium-on CXL cornea with minimal appearance of the demarcation line. Note: Of the 61,770 pixels (cornea cross-section), only 443 correspond to a hyper-reflective area.Abbreviation: CXL, cross-linking.

Figure 5 Example of corneal cross-sectional images examined in the study showing various degrees of demarcation line extent.


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by 0.02% per month), indicating that the demarcation line area fades away by postoperative month 12, in agreement with our clinical findings.15

The depth of the demarcation line, found to be on average of 305 m, is consistent with the accepted notion that in order to avoid ultraviolet-A irradiation damage to the corneal endothelium,23 the CXL parameters are set in a way that effective treatment occurs only in the first 300 m of the corneal stroma.12

The depth of the demarcation line appears, on the other hand, to be stable over time, even after 3 years following operation, at approximately 70% of the corneal depth. However, a deeper demarcation line depth (relative to the corneal depth) is associated with thinner corneal thickness, as measured postoperatively. In the selected 12 thinner corneas, the depth of the demarcation line was found to be 83% of the total corneal thickness.

One clinical example of ineffective CXL action is demonstrated in Figure 4, in which a case of a cornea treated in another institution with epithelium-on CXL technique demonstrated minimal signs of hyper-reflective areas.24 This case, which was not part of the case study, was presented to our practice with progressive ectasia following the CXL operation in another practice. Examples of corneal cross-sectional images examined in the study showing various degrees of demarcation line extent are presented in Figure 5.

ConclusionAS-OCT appears to demonstrate reproducible early (1 month) and long-term (up to 3 years) CXL cornea findings. The hyper-reflective lines may represent induced cornea density changes or subtle intrastromal cornea scarring. This novel quantitative and qualitative technique may constitute a possible benchmark for a noninvasive measurement to evaluate and titrate the amount, extent, and depth of intrastromal effects of the CXL treatment in KCN and possibly ectasia eyes.

DisclosureThe authors report no conflicts of interest in this work.

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