A Comparison of Three Methods of Measuring Central Corneal Thickness in Normal and Thinned Corneas

A COMPARISON OF THREE METHODS OF MEASURING CENTRAL CORNEAL THICKNESS IN NORMAL AND THINNED CORNEAS

A Thesis

Presented in Partial Fulfillment of the Requirements for

The Degree of Master of Science in the

Graduate School of The Ohio State University

Amber J. Colling, B.A. Graduate Program in Vision Science

* * * * *

The Ohio State University 2010

Master’s Examination Committee:

Dr. Mark Bullimore, MCOptom, PhD, Advisor

Dr. Jeffrey Walline, OD, PhD

Dr. Karla Zadnik, OD, PhD

Amber J. Colling

2010

ABSTRACT

The thickness of the cornea is an important parameter in Goldmann applanation

tonometry, corneal disease, contact lens wear, and refractive surgery. Pachymetry is the

measurement of corneal thickness, and currently ultrasound pachymetry is the gold

standard. Several non-contact instruments, the Orbscan II (Orbtek, Inc.) and the Visante

anterior segment-optical coherence tomographer (Carl Zeiss Meditec) have been

introduced that provide more information about the cornea including corneal thickness

topography maps. Both of these give corneal thickness measurements across the entire

surface of the cornea in contrast to ultrasound pachymetry, which only provides

information at one point and requires topical anesthetic and corneal contact.

Each of these instruments is based on different principles. Ultrasound is based on

the reflection of sound from the anterior and posterior corneal surfaces The Orbscan II is

a corneal topographer that measures corneal thickness by analyzing reflections from the reflecting surfaces of the cornea via slit-scanning technology and videokeratography. The

Visante AS-OCT uses a Michelson-type interferometer where scanning infrared spots move across the corneal surface with multiple axial scans to form high-resolution cross- sectional images. The technology was first applied in ophthalmic use as retinal OCT.

The anterior segment OCT uses a longer wavelength of 1310 nm versus 820 nm of the

retinal OCT allowing for better penetration through tissues.

Little information is available comparing the Ultrasound, Orbscan II and the

Visante in normal corneas and thin corneas. The goal of the present study is to compare central corneal thickness measurements from three instruments in normal corneas and corneas that have been thinned due to refractive surgery or keratoconus. To our knowledge, this is the first study to examine central corneal thickness measured by these three instruments in normal and thinned corneas. The aim was to determine repeatability of the instruments, to assess agreement between the instruments, and to assess the effect of corneal thickness on repeatability and agreement. Because each instrument is based on different principles, measurements may be biased when normal versus abnormally thin corneas are measured.

Forty-five subjects (15 normal, 15 post-LASIK, and 15 keratoconus) were examined on two occasions, separated by one-to-seven days. Three measurements (later averaged) were made on the right eye of each subject using the three techniques. The subjects were analyzed as three groups: normal corneas, post-LASIK and keratoconus.

The data were entered into a Microsoft Excel spreadsheet and were analyzed using SPSS statistical software. Mixed model analysis of variance was used to compare between instruments, visit, and within groups. For each technique and cornea type, the difference

between instruments and visits was determined for each subject and analyzed using

Bland-Altman analysis to produce the mean, standard deviation, and 95% limits of

agreement (LoA). A paired two-sample t test was also conducted for validity and

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repeatability. For normal corneas, mean central corneal thickness was as follows: 550 ±

34 μm for ultrasound, 541 ± 44 μm for Orbscan II, and 532 ± 38 μm for Visante. For post-LASIK corneas, mean central corneal thickness was as follows: 502 ± 40 μm for ultrasound, 495 ± 52 μm for Orbscan II, and 485 ± 42 μm for Visante. For keratoconic corneas mean central corneal thickness was as follows: 529 ± 46 μm for ultrasound, 498

± 44 μm for Visante, and 490 ± 53 μm for Orbscan II. Mixed model analysis showed a significant interaction between instrument and cornea types (p=0.012). That is, the instruments not only measured significantly different from each other (p < 0.001) but also significantly different when measuring the various cornea types. Repeatability was poorer for all techniques in the keratoconic corneas and for ultrasound. For normal corneas, the

Visante was the most repeatable: 95% LoA of –8 to +8 μm followed by the Orbscan:

95% LoA of –11 to +12 μm, and ultrasound: 95% LoA of –15 to +19 μm. For post-

LASIK corneas, the Orbscan was the most repeatable: 95% LoA –11 to +11 μm followed by the Visante: 95% LoA of –10 to +13, and ultrasound: 95% LoA –24 to +16 μm. For keratoconic corneas, the Visante was the most repeatable: 95% LoA –36 to +37 μm followed by the Orbscan: 95% LoA –31 to +47 μm and ultrasound: 95% LoA –40 to +53

μm.

The results of this study show that there was a significant interaction between the different instruments and the types of corneas that were measured. The Orbscan II and

Visante underestimate corneal thickness compared to the ultrasound in normal and post-

LASIK corneas. The ultrasound did give a higher thickness than the Visante and the

Orbscan II in keratoconic corneas; however the Visante gave a higher central corneal iv

thickness than the Orbscan II. Though ultrasound has been considered the gold standard,

its limitations have long been recognized including the need for topical anesthetic and

subsequent contact with a probe. It only yields a single point measurement of which the accuracy and repeatability may be affected by inappropriate probe positioning. The optical techniques provide more information about the entire cornea and do not require contact. The data acquired by these techniques can be invaluable to clinicians, enabling them to detect sub-clinical keratoconus, follow the progression of disease, and assess pre- and post- keratorefractive and keratoplasty outcomes. It is important to note that in regards to pachymetry, central corneal thickness acquired by the Visante and Orbscan are

not yet considered interchangeable with ultrasound.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my co-advisors Drs. Mark Bullimore and

Kathryn Richdale. Dr. Bullimore gave me the opportunity to do research. His belief in me gave me the confidence to expand on my T35 summer research project and pursue the

Master of Science. This accomplishment means more to me than he may ever know! Dr.

Richdale has been my ever-supportive mentor and friend. She is an exemplary clinician and researcher, and I am forever indebted to her for her unwavering guidance and support. I would also like to thank Drs. Jeffrey Walline and Karla Zadnik for their time and effort spent in reviewing and enhancing this study. Their encouragement motivated me to continue the course and achieve the goal!

I would also like to thank my parents and my sister for their never-failing love, prayers, and support. They have always taught me to reach higher and never settle for mediocre. And last but not least, I thank Landon Colling for his love and patience. He has always encouraged me to follow my dreams and never give up.

VITA

April 22, 1983 Born – Olathe, Kansas

2004 B.A. Biology, Olivet Nazarene University

2010 Doctor of Optometry, The Ohio State University

FIELDS OF STUDY

Major Field: Vision Science

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TABLE OF CONTENTS

Abstract ii Acknowledgements vi Vita vii List of Figures x List of Tables xii

Chapters:

1. Historical Review 1.1 Introduction 1 1.2 Corneal Anatomy 3 1.3 Factors Influencing Corneal Thickness 4 1.3.1 Age, Gender, Ethnicity 4 1.3.2 Keratoconus 5 1.3.3 Refractive Surgery 8 1.4 Pachymetry 10 1.4.1 Ultrasound 10 1.4.2 Orbscan II 11 1.4.3 Visante 11 1.5 Comparative Studies 12 1.5.1 Studies of Normal Corneas 12 1.5.2 Studies of Post-LASIK Corneas 14 1.5.3 Studies of Keratoconic Corneas 14 1.6 Repeatability Studies 15 1.7 Summary 15

2. Objectives 20

3. Methods 3.1 Overview 21 3.2 Subjects 21 3.3 Instruments 22 3.4 Summary of Study Protocol 22 3.5 Statistical Methods 24

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4. Results 4.1 Overview 26 4.2 Mean Central Corneal Thickness 26 4.3 Between Session Repeatability 29

5. Discussion 5.1 Overview 43 5.2 Central Corneal Thickness 44 5.3 Repeatability 45 5.4 Ultrasound Pachymetry 46 5.5 Orbscan Pachymetry 47 5.6 Visante Pachymetry 50 5.7 Summary 51 5.8 Limitations 52

6. Conclusions 57

Appendix: Tables of Individual Subject Data 59

List of References 105

LIST OF FIGURES

Figure 1: Picture of ultrasound pachymeter 17 Figure 2: Picture of Orbscan topographer and pachymetry map 18 Figure 3: Picture of Visante AS-OCT with anterior segment picture and pachymetry map 19 Figure 4: Results comparing mean central corneal thickness in each of the three subject groups and for each of the three techniques 34 Figure 5: Validity: Visante versus Ultrasound for all cornea types. 35 Figure 6: Validity: Orbscan versus Ultrasound for all cornea types 35 Figure 7: Validity: Visante versus Orbscan for all cornea types 35 Figure 8: Validity: Visante versus Ultrasound for normal corneas 36 Figure 9: Validity: Orbscan versus Ultrasound for normal corneas 36 Figure 10: Validity: Visante versus Orbscan for normal corneas 36 Figure 11: Validity: Visante versus Ultrasound for post-LASIK corneas 37 Figure 12: Validity: Orbscan versus Ultrasound for post-LASIK corneas 37 Figure 13: Validity: Visante versus Orbscan for post-LASIK corneas 37 Figure 14: Validity: Visante versus Ultrasound for keratoconic corneas 38 Figure 15: Validity: Orbscan versus Ultrasound for keratoconic corneas 38 Figure 16: Validity: Visante versus Orbscan for keratoconic corneas 38 Figure 17: Repeatability of Visante for all cornea types 39 Figure 18: Repeatability of Orbscan for all cornea types 39 Figure 19: Repeatability of Ultrasound for all cornea types 39 Figure 20: Repeatability of Visante for normal corneas 40 Figure 21: Repeatability of Orbscan for normal corneas 40 Figure 22: Repeatability of Ultrasound for normal corneas 40 x

Figure 23: Repeatability of Visante for post-LASIK corneas 41 Figure 24: Repeatability of Orbscan for post-LASIK corneas 41 Figure 25: Repeatability of Ultrasound for post-LASIK corneas 41 Figure 26: Repeatability of Visante for keratoconic corneas 42 Figure 27: Repeatability of Orbscan for keratoconic corneas 42 Figure 28: Repeatability of Ultrasound for keratoconic corneas 42

LIST OF TABLES

Table 1: Mean central corneal thickness for each group 31 Table 2: Validity for each instrument and cornea type 32 Table 3: Repeatability for each instrument and cornea type 33 Table 4: Studies comparing normal corneas 53 Table 5: Studies comparing post-LASIK corneas 54 Table 6: Studies comparing keratoconic corneas 55 Table 7: Studies comparing repeatability in normal corneas 56

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CHAPTER 1

HISTORICAL REVIEW

1.1 Introduction

Corneal thickness is a valuable indicator of the health and physiology of the cornea. Specifically, central corneal thickness (CCT) measurements are vitally important for the diagnosis, treatment, and management of various ocular conditions. For instance, central corneal thickness is not only a key risk factor for the development of glaucoma but also affects Goldmann applanation tonometry, a critical parameter of the treatment of glaucoma (Kass, Heuer et al. 2002). Central corneal thickness is also important in contact lens research. Central corneal thickness allows the extent of corneal edema to be determined in order to ensure the level of safety needed for extended contact lens wear

(Hashemi and Mehravaran 2007). Central corneal thickness is essential information in situations where the cornea is thinned, either pathologically as in the case of keratoconus or intentionally via refractive surgery. In refractive surgery, central corneal thickness is a crucial determinant of the amount of treatment that needs to occur for the desired refractive outcome and also for the avoidance of postoperative keratorefractive complications (Fakhry, Artola et al. 2002).

Pachymetry, the measurement of corneal thickness, was first performed in the

1600’s (Cairns and McGhee 2005). Ultrasound pachymetry is currently the gold standard,

however it requires the use of topical anesthetic and contact with the cornea. It is based

on the reflection of sound from the anterior and posterior corneal surfaces although the exact posterior corneal reflection point for ultrasound waves is not known (Ehlers, Shah et al. 2008). Several non-contact instruments, the Orbscan II and the Visante anterior segment-optical coherence tomographer (AS-OCT) have been introduced that provide more information about the cornea including corneal thickness topography maps. Both of these give corneal thickness measurements across the entire surface of the cornea in contrast to ultrasound pachymetry, which provides information at one point. Despite the different techniques, it is important that each pachymeter has the ability to produce similar results on different occasions. This refers to the repeatability of an instrument, which is important because it allows the clinician to more accurately detect central corneal thickness changes over time.

Little information is available comparing the Ultrasound, Orbscan II and the

Visante in normal corneas and thin corneas. The goal of the present study is to compare central corneal thickness measurements from three instruments in normal corneas and corneas that have been thinned due to refractive surgery or keratoconus. To our knowledge, this is the first study to examine central corneal thickness measured by these three instruments in normal and thin corneas. The aim was to determine repeatability of the instruments, to assess agreement between the instruments, and to assess the effect of corneal thickness on repeatability and agreement. Because each instrument is based on

different principles, measurements may be biased when normal versus abnormally thin corneas are measured.

1.2 Corneal anatomy

The cornea is a viscoelastic tissue made up of 5 layers including the epithelium,

Bowman’s layer, stroma, Descemet’s membrane and the endothelium. The epithelium is stratified, squamous, and non-keratinized and about 50 μm thick. It acts as a biodefense system and provides an optically smooth refracting surface. Bowman’s layer is an acellular superficial layer of the stroma. The stroma makes up about 90% of the cornea. It is primarily composed of regularly oriented layers of collagen, where the spacing is maintained by proteoglycan ground substance including chondroitin sulphate and keratan sulphate interspersed with keratocytes. The thickness of the cornea is affected by its hydration, where a linear relationship exists between its thickness and the hydration of the stroma. In the presence of stromal edema, the regular spacing of the collagen fibers is disturbed leading to light scatter and corneal haze. Descemet’s membrane is the basement membrane of the endothelium and consists of a fine latticework of collagen fibrils. It contains an anterior banded zone that is deposited in-utero, and a posterior non-banded zone that is laid down throughout life by the endothelium. The endothelium is a single layer of hexagonal cells that do not regenerate. It plays a crucial role in maintaining corneal deturgescence. The adult cell density is about 2500 cells/mm2; the number of

cells decreased at about 0.6% per year. Corneal edema develops resulting in reduced

corneal transparency when the cell density decreases to about 500 cells/mm2 (Krachmer,

Mannis et al. 2005).

1.3 Factors influencing corneal thickness

Because central corneal thickness is a dynamic biometric variable, it is important

to understand its normal fluctuations in order to more effectively interpret temporary or

long-term induced or pathologic changes. Doughty and Zaman conducted a meta-analysis

of 230 data sets in which inter-individual variance was reported and found a group-

averaged central corneal thickness of 536± 31μm for normal corneas (Doughty and

Zaman 2000). Many factors can influence the range of central corneal thickness values

including regional differences in the corneal thickness profile, diurnal variation, contact

lens wear, pregnancy, topical ophthalmic medications, and systemic disease (Weinreb, Lu

et al. 1988; Doughty and Zaman 2000). These changes, however, fall within the expected

variance of central corneal thickness. In contrast, corneal pathology and ocular surgery

produce changes in central corneal thickness that not only merit monitoring it but also

may affect the very methods for measuring corneal thickness.

1.3.1 Age, gender, ethnicity

While there is no obvious age-dependent change in central corneal thickness for

eyes of Caucasians, there is a clinically relevant age-dependent decline in central corneal

thickness after age 60 in non-white eyes of 3 to 7 μm/decade (Alsbirk 1978; Doughty and

Zaman 2000). Hahn et al. found that central corneal thickness in Latinos was less than that in Caucasians but greater than that in African Americans and Asians. Black, Asian,

Latino, Greenland Eskimo, and Native American corneas are possibly thinner than

Caucasian corneas (Hahn, Azen et al. 2003). It is difficult to compare the eyes of

different races because of the differing prevalence of conditions that cause corneal

thickness changes such as diabetes (Doughty and Zaman 2000). Also, past studies have

not included adequate representation from different racial or ethnic groups.

In Doughty’s meta-analysis, a gender difference of central corneal thickness was

determined. The over-all average values reported for all-women studies, which was 554

μm, is slightly higher than the average value of 535 μm. Several factors could account for

the increased thickness including hormonal changes, use of oral contraceptives, and pregnancy (Soni 1980; Weinreb, Lu et al. 1987; Weinreb, Lu et al. 1988).

1.3.2 Keratoconus

Keratoconus is a progressive, non-inflammatory ectasia of the cornea

characterized by corneal steepening, apical thinning, and central scarring (Wagner, Barr

et al. 2007). It typically manifests in adolescence with variable rate of progression until

the third or fourth decades of life of years where it then tends to arrest. It is bilateral in

around 90% of cases; however, it appears that severe disease presents more

asymmetrically. Other features of keratoconus include irregular astigmatism, high

myopia, and visual distortions with mild to marked visual impairment (Ehlers, Shah et al.

2008).

The diagnosis of keratoconus can be made utilizing various diagnostic tools such

as biomicroscopy, keratometry, pachymetry, and computer-assisted topography. The

most sensitive method of detecting early keratoconus is corneal topography, where

irregular astigmatism can be visualized. It is also the most useful for monitoring the

5 progression and subsequent development of the central or paracentral stromal thinning and apical protrusion (Kanski 2007).

Slit lamp biomicroscopy may also reveal characteristic signs including the ectatic protrusion of the cornea, “oil-drop” reflex, corneal scarring, Munson’s sign, Vogt’s vertical striae, and a Fleischer ring. Munson’s sign is a lower lid bulge on down gaze, whereas Vogt’s vertical striae are areas of stressed collagen lamella located in the stroma that tend to radiate from the center of the cone. Fleischer’s ring is an iron deposit in the epithelium circumferentially around the base of the cone. Ultrasound pachymetry can also enable the clinician to measure and track the progression of corneal thinning over time.

The etiology of keratoconus remains unclear, though it has been associated with multiple ocular and systemic associations. Ocular associations include vernal keratoconjunctivitis, Fuchs’ dystrophy, pellucid marginal corneal degeneration, aniridia, blue sclera, Leber’s congenital amaurosis, retinitis pigmentosa. Systemic associations include Down syndrome, Ehlers-Danlos syndrome, Marfan’s syndrome, Turner syndrome, mitral valve prolapse, osteogenesis imperfecta, and atopy. It is interesting to note that while there have been many associations of keratoconus with other connective tissue disease, the CLEK study found that keratoconus is not associated with an increased risk of connective tissue disease (Wagner, Barr et al. 2007). The role of heredity has not been well defined. The frequency of inheritance has been estimated to be about 6%, where autosomal dominant with incomplete penetrance, recessive inheritance, and

isolated gene mutation has been proposed modes of inheritance patterns (Krachmer,

Mannis et al. 2005).

While the various ocular and systemic associations have led to the development of

many hypotheses regarding the etiology of keratoconus, the cause remains an enigma.

Studies in biochemistry have provided some insight, yet still remain inconclusive. It has been shown that the total amount of corneal protein is decreased in keratoconus, and that the level of degradative lysosomal enzymes and some matrix metalloproteinases are elevated. It has also been shown that keratocytes from keratoconus corneas have four times the interleukin-1 binding sites compared to normal corneas. Interleukin-1 has induced apoptosis of stromal keratocytes in vitro, and apoptosis has been found in the stromal keratocytes of keratoconus corneas but not in normal control corneas. Thus, keratocytes in keratoconus corneas may have increased sensitivity to the effects of interleukin-1 (Krachmer, Mannis et al. 2005).

The treatment of keratoconus includes spectacles in early cases to correct regular and mildly irregular astigmatism. Contact lenses are the next form of treatment when glasses fail to provide adequate visual function. Contact lens options include soft toric lenses, rigid gas-permeable (RGPs) lenses, piggyback soft and RGP lenses, and scleral lenses. The mainstay of treatment has been relatively flat-fitting RGPs with light apical corneal touch, also known as the “three point touch” technique. There is evidence to suggest that the risk of corneal scarring in contact lens wearers is higher than in non- contact lens wearers. A randomized clinical trial would need to be conducted to determine if a casual relationship exists between certain fitting philosophies of RGPs and

corneal scarring (Wagner, Barr et al. 2007). Keratoplasty is the next form of treatment in

patients with advanced disease. Keratoplasty is indicated for corneas with significant

scarring or when the patient becomes contact lens intolerant. Though clear grafts are obtained in 85% of cases, residual astigmatism and anisometropia may necessitate the

need to initiate or continue contact lens wear (Kanski 2007).

1.3.3 Refractive Surgery

Refractive surgery procedures are used to remodel different ocular structures to

improve the refractive state of the eye and decrease or eliminate dependency on glasses

or contact lenses. Various methods of surgical correction are employed and overall can be

categorized as corneal or lenticular procedures. Keratorefractive procedures can be

divided into corneal incision procedures and flap and surface procedures. Corneal

incision procedures include radial keratotomy, astigmatic keratotomy, and limbal

relaxing incisions. Flap and surface procedures use photoablation and include

photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), and laser

subepithelial keratomileusis (LASEK). These procedures have a greater effect on central

corneal thickness. Less commonly used keratorefractive procedures include

epikeratoplasty, implantation of plastic intrastromal corneal ring segments, keratophakia, and laser thermal keratoplasty. Lenticular refractive procedures include cataract surgery and clear lens extraction with intraocular lens implantation (IOL), phakic IOL implantation, accommodative IOL implantation, and piggyback IOL implantation .

Currently there are three major surgical techniques that use a 193 nm argon- fluoride excimer laser to ablate the cornea for a desired refractive outcome. These corneal

thinning techniques can further be divided into flap procedures or surface procedures.

LASIK, more widely performed, is considered a flap procedure because the surgeon uses

an instrument called a microkeratome or a femtosecond laser to create a flap of corneal

tissue. The flap, which is usually 100-180 μm thick, is then lifted like a hinge door so that a predetermined amount of corneal stromal tissue can be ablated with the excimer laser.

Our study included patients who had undergone LASIK, which is indicated for spherical refractive range of -10.00 to +4.00 D with up to 4.0 D of astigmatism. PRK and LASEK are considered surface procedures, where the main difference is in the handling of the epithelium. Surface procedures do not require a partial thickness cut into the stroma as in

LASIK. In PRK, the epithelium is removed so that a flap is not created. Stromal ablation is performed as done in LASIK, however, a bandage contact lens is used. Recovery time

is longer in PRK than in LASIK, lasting up to 3 months or longer. In LASEK, the

epithelium is loosened with an alcohol solution and then removed with a trephine blade.

Again, stromal ablation is performed using the excimer laser.

In all three procedures, central corneal thickness is vitally important because it

allows the surgeon to determine if the patient has enough tissue to ablate for the desired

refractive outcome and avoidance postoperative complications. The formula used to

determine a safe amount of residual thickness is as follows:

CCT-thickness of flap–depth of ablation=residual bed thickness. About 15 μm of tissue is

ablated per diopter of refractive error. Most surgeons require a stromal bed reserve of 250

μm after the tissue has been ablated. This amount has been shown to be necessary for

corneal stability and the prevention of corneal ectasia after LASIK (Sonmez, Doan et al.

2007). PRK and LASEK procedures are considered over LASIK in patients with thinner

corneas because less corneal tissue is required when a flap is not created.

1.4 Pachymetry

Pachymetry can be conducted using various methods including ultrasound or

optical techniques. Conventionally, optical pachymetry refers to slit lamp based optical

pachymetry but can also refer to all techniques that involve the use of light such as specular microscopy, spectral oscillation interferometry, confocal microscopy, optical coherence tomography, and slit scanning topography. There are various advantages and

disadvantages of the various techniques. Ultrasound is portable and typically less

expensive than the other instruments, however, it requires contact with the cornea making

it more invasive. Optical techniques do not require corneal contacts thus are less invasive

but may be affected by various corneal conditions that cause light scatter or absorption.

1.4.1 Ultrasound

Ultrasound is currently the gold standard and is based on the speed of sound. The time delay between an ultrasound emitted pulse and the reflected echo from an acoustic interface is directly related to the thickness of the tissue, although the exact posterior corneal reflection point is not known (Ehlers, Shah et al. 2008). The velocity of sound

through the media is important, where the echoes result from changes in the speed of

sound within the tissue. The following formula depicts how thickness measurements can

be derived: ΔT=Δz/v where ΔT is the echo delay, Δz is the distance that the echo travels, and v is the velocity (Thornton 1986). See figure 1.

1.4.2 Orbscan II

The Orbscan II is a corneal topographer that measures corneal thickness by analyzing reflections from the reflecting surfaces of the cornea via slit-scanning technology and videokeratography. A total of 40 slits are projected onto the cornea at a

45o angle, the images of which are captured and then used to reconstruct the anterior and posterior surfaces (Marsich and Bullimore 2000). The anterior surface elevation is subtracted from the posterior surface elevation obtain the corneal thickness. Corneal thickness is averaged in nine circles of 2 mm located in the center of the cornea and eight locations in the mid-periphery that are located 3 mm from the visual axis (Liu, Huang et al. 1999). See figure 2.

1.4.3 Visante

The Visante uses a Michelson-type interferometer to measure the delay and intensity of backscattered light by comparing it to light that has traveled a known reference path length and time delay. Scanning infrared spots move across the corneal surface with multiple axial scans to form high-resolution cross-sectional images. The global pachymetry map uses 10 mm radial lines in 16 equally spaced meridians centered on the corneal vertex. Each radial line is composed of 128 A-scans, where the entire map contains 2,048 A-scans acquired in 0.5 seconds. A pachymetry map is generated automatically and divided into four zones including: central 0-2mm, pericentral 2-5 mm, transitional 5-7 mm, and peripheral 7-10 mm zones. These are further divided into eight sectors in between eight radial lines. See figure 3. The technology was first applied in ophthalmic use as retinal OCT. The anterior segment OCT uses a longer wavelength of

1310 nm versus 820 nm of the retinal OCT allowing for better penetration through tissues that produce high light scatter such as the sclera and limbus (Konstantopoulos, Hossain et al. 2007). See figure 3.

1.5 Comparative Studies

Little information is available comparing the Ultrasound, Orbscan II and the

Visante in normal corneas and thinned corneas. Because the Visante is relatively new, very few studies have been conducted with it. To our knowledge, this is the first study to determine the validity of central corneal thickness measurements by these three instruments in normal and thin corneas. Refer to tables 4 through 6 for a comparison of different study findings.

1.5.1 Studies of Normal Corneas

Early comparisons of ultrasound and Orbscan in normal corneas demonstrated that the Orbscan overestimated central corneal thickness (Yaylali, Kaufman et al. 1997;

Chakrabarti, Craig et al. 2001; Gherghel, Hosking et al. 2004; Fishman, Pons et al. 2005;

Doughty and Jonuscheit 2007; Hashemi, Roshani et al. 2007). Yaylali et al. reported an overestimation by 23 to 28 μm (Yaylali, Kaufman et al. 1997). It has been suggested that the source of discrepancy could be due to the Orbscan measuring the tear film. King-

Smith reported a tear film measurement of 2.7 μm (King-Smith, Fink et al. 2004). Thus, it has been proposed that other errors must lead to the discrepancy (Gonzalez-Meijome,

Cervino et al. 2003). In contrast, the tear film is said to be displaced upon applanation of the ultrasound probe therefore is not included in the measurement. It has been reported that on average, scanning-slit topography yields 9.6% higher readings than ultrasound

12 pachymetry. In response to this discrepancy, the manufacturer recommended a 0.92 acoustic equivalent correction factor, which reduces the readings by 8% (Suzuki, Oshika et al. 2003). Studies that have been conducted using ultrasound and the Orbscan II on normal corneas find similar central corneal thickness values when an acoustic correction factor is applied. Many of the studies have found that ultrasound gives slightly higher central corneal thickness values compared to Orbscan II though these are not statistically significant (Gonzalez-Meijome, Cervino et al. 2003; Suzuki, Oshika et al. 2003; Amano,

Honda et al. 2006; Haque, Simpson et al. 2006). McLaren et al., however, did find that ultrasound measured statistically higher than Orbscan II (McLaren, Nau et al. 2004;

Hashemi and Mehravaran 2007). Marsich and Bullimore found a statistically higher value given by Orbscan (Marsich and Bullimore 2000).

Less data are available comparing central corneal thickness measures derived from the Visante. Zhao et al. and Kim et al. found that the ultrasound measured statistically higher than the Visante in normal corneas (Zhao, Wong et al. 2007; Kim,

Budenz et al. 2008). Li et al. compared ultrasound, the Orbscan II and the Visante in normal corneas and found that ultrasound and Orbscan II did not yield statistically different values when using a correction factor of 0.89. They did, however, find that the

Visante measured significantly lower compared to both ultrasound and the Orbscan II

(Li, Mohamed et al. 2007). It is unclear at this time the source of discrepancy between ultrasound and Visante measurements. It is has been proposed that for OCT, uncertainty of the true index of refraction of infrared radiation in the cornea creates a source of error in calculating the central corneal thickness (Kim, Budenz et al. 2008).

1.5.2 Studies of Post-LASIK Corneas

There have been several studies conducted to determine the validity of corneal thickness measurements acquired by ultrasound and Orbscan in post-refractive surgery cases. Hashemi and Kawana et al. found that the Orbscan II significantly underestimated central corneal thickness in post-LASIK corneas as compared to ultrasound (Kawana,

Tokunaga et al. 2004; Hashemi and Mehravaran 2007). Fakhry et al. however, found no statistically significant difference (Fakhry, Artola et al. 2002). Ho et al. looked at central corneal thickness measurements in post-LASIK corneas obtained via ultrasound, Orbscan

II and Visante. It was determined that Visante measurements significantly underestimated corneal thickness, whereas ultrasound and Orbscan II were not significantly different

(Ho, Cheng et al. 2007). Cheng et al. found that the Orbscan II and the Visante both measured significantly lower than ultrasound after LASIK. The Visante had better agreement and correlation with ultrasound than with Orbscan II (Cheng, Rao et al. 2008).

1.5.3 Studies of Keratoconic Corneas

Because keratoconus is a degenerative disease of the cornea that results in thinning and curvature changes, studies have been conducted to determine corneal thickness in these pathological cases. Central corneal thickness in keratoconic corneas has previously been reported using Orbscan and ultrasound, but no studies have compared these to the Visante. Haque et al. and Gherghel et al. reported that the Orbscan

II yielded significantly lower central corneal thickness values than ultrasound (Haque,

Simpson et al. 2006). Interestingly, Gherghel et al. also determined that Orbscan II values

without the correction factor were lower than ultrasound, however not statistically

significant (Gherghel, Hosking et al. 2004).

1.6 Repeatability Studies

Little information is available comparing the repeatability of Ultrasound, Orbscan

II and the Visante in normal corneas and thinned corneas. Suzuki et al. compared same-

day repeatability of ultrasound and the Orbscan II in normal corneas and found that the

Orbscan II was slightly more repeatable but the difference was not statistically significant

(Suzuki, Oshika et al. 2003). Similarly, Marsich and Bullimore found better repeatability of the Orbscan versus ultrasound (Marsich and Bullimore 2000). Amano et al. compared the repeatability of ultrasound and the Orbscan II and found that ultrasound was more repeatable than the Orbscan II. They proposed that an experienced examiner could obtain more repeatable results using the ultrasound and that patient fixation can affect Orbscan measurements (Amano, Honda et al. 2006). Maldonado et al. assessed the repeatability of the Orbscan II in post-LASIK corneas and found good intersession repeatability

(Maldonado, Lopez-Miguel et al. 2009). Mohamed et al. determined excellent repeatability in both normal and keratoconus central corneal thickness acquired by the

Visante (Mohamed, Lee et al. 2007). Refer to table 7 for a comparison of repeatability findings.

1.7 Summary

Central corneal thickness is a dynamic variable that can be affected by many factors including ocular pathology and ocular surgery. Thus, it is an important indicator of corneal physiology and stability. There are several techniques available to measure

central corneal thickness, though there are reported discrepancies in normal as well as

thinned corneal values. Ultrasound pachymetry is more often clinically used, however,

with the introduction of newer technologies it is prudent to determine their validity and repeatability as compared to ultrasound, the gold standard. It is also important to determine possible explanations for the existence of bias between the different techniques when normal and thinned corneas are measured. The purpose of this study was to determine repeatability of ultrasound, the Orbscan II and the Visante, to assess agreement between the instruments, and to assess the effect of corneal thickness on repeatability and agreement. Because each instrument is based on different principles, measurements may be biased when normal versus abnormally thin corneas are measured. To our knowledge, this is the first study to assess the validity and repeatability of these three instruments in normal, post-Lasik, and keratoconic corneas.

Figure 1. Picture of ultrasound pachymeter

Figure 2. Picture of Orbscan topographer and pachymetry map

Figure 3. Picture of Visante AS-OCT with anterior segment picture and pachymetry map 19

CHAPTER 2

OBJECTIVES

In this study, the central corneal thickness (CCT) was measured on the right eye of forty-five subjects including fifteen with normal corneas, fifteen with post-LASIK corneas, and fifteen with keratoconus corneas. Measurements were taken using ultrasound pachymetry, the Orbscan II topographical system, and the Visante Anterior

Segment optical coherence tomographer. The study had the following objectives:

• To determine the repeatability of the three instruments stated above for measuring

central corneal thickness on two separate days

• To determine the validity of central corneal thickness measurements made by the

Orbscan II and the Visante as compared to ultrasound, the clinical gold standard

• To determine if bias exists between the instruments when measuring normal or

thinned corneas

CHAPTER 3

METHODS

3.1 Overview

Central corneal thickness (CCT) was measured on the right eyes of fifteen normal subjects, fifteen post-LASIK subjects, and fifteen keratoconus patients using three different instruments. Three measurements were taken by each instrument on two occasions separated by one to fourteen days. Statistical analyses were performed to

compare the agreement and repeatability between instruments, types of corneas, and

visits.

3.2 Subjects

Forty-seven subjects enrolled in the study, and forty-five completed the study because two subjects were lost to follow-up. There were twenty-five females and twenty males of Caucasian and African American heritage. The age of the subjects ranged from

21 to 72 years old. The range of the duration post-surgery in the LASIK corneas was 1 year from surgery to 10 years post-surgery with a mean of 5 years. The range of the duration of keratoconus was 1 year to 55 years with a mean of 24 years. Subjects with normal and post-LASIK corneas were free of contact lens wear at least twelve hours

before the first examination through the end of the second examination. Pregnant women

were excluded from the study.

Patients were educated on the purpose of the study and informed consent was

obtained from each patient before beginning the examination. The Ohio State

University’s institutional review board, in accordance with the Declaration of Helsinki,

approved the study protocol.

3.3 Instruments

Central corneal thickness was measured using three instruments:

1. The Cornea-gauge Plus ultrasonic pachymeter (Sonogage);

2. The Orbscan II (Bausch & Lomb), a slit-scan topographer; and

3. The Visante (AS-OCT, Carl Zeiss Meditec, Inc.)

3.4 Summary of Study Protocol

Once the subjects met inclusion criteria, they participated in two measurement sessions. At the first session, subjects provided information regarding age, gender, race and ethnicity. Post-LASIK subjects were asked to provide the date of their refractive surgery, while keratoconus patients were asked for the date their diagnosis. Central corneal thickness of the right eyes was measured with the three instruments at each visit.

Ultrasound pachymetry was performed last because it requires contact with the cornea.

Three measurements were taken at each visit via the Orbscan and Visante. Five measurements were taken with the ultrasonic pachymeter as recommended by the manufacturer; three of the measurements were included in the data analysis. Central

corneal thickness measurements were repeated at a second session one to fourteen days

later.

One measurement of refractive error and keratometry was taken with an auto-

refractor/auto-keratometer at the initial visit. Keratoconus patients were instructed to

remove their contact lenses at this point. The patient was positioned in the chin and

forehead rest so that the measurement of the right eye could be acquired. The patient was

instructed to look straight at the fixation target. The exam mode was set to automatic, and

the joystick was moved to align the eye. When alignment was achieved, the button was

pressed to acquire the measurement. The measurements were recorded in the patient

charts.

Three Visante measurements were made at each visit. Each patient’s initials,

study number, and refractive error determined by the autorefractor were entered into the

Visante computer. The left eye was patched, and the patient was situated properly on the

chin and forehead rest. The patient was instructed to look straight ahead at the fixation

target. Once the cornea was aligned on the computer screen, a vertical line would pass

through the center of the cornea on the screen. The pachymetry map was displayed on the

computer screen. Two more subsequent measurements were then taken. Three Orbscan

measurements were made at each visit. The left eye was patched and the subject was

properly situated on the chin and forehead rest. The image of the placido disk was

focused on the cornea, and the visible half slits were aligned one over the other to form an elongated “S.” The subject was instructed to hold the eyes open while the acquisition button was pressed and the scan acquired. After two more subsequent scans were

23 acquired, maps of all three scans were generated. For ultrasound pachymetry, a set of five measurements was taken on each subject at each visit. A drop of proparacaine ophthalmic solution was put in the right eye of each subject. The subject was then instructed to fixate on a distance target. The cornea of the right eye was contacted centrally with the probe.

The probe was removed from the cornea between each reading. Applanation was performed until five measurements were made, as suggested by the manufacturer. The first three measurements were used for data analysis. In cases where any of the first three numbers appeared inconsistently higher or lower, the three most similar numbers were used for analysis. See the appendix for demographics and raw pachymetry data for all subjects.

3.5 Statistical Methods

The data were entered into a Microsoft Excel spreadsheet and were analyzed using SPSS statistical software. To assess validity, mixed model analysis of variance was used to compare between instruments, visit and within groups. Because an interaction was found, Bland-Altman analysis and a paired two-sample t test were then conducted to determine which set of factors drove the interaction found in the mixed model analysis of variance. For each subject and instrument, three measurements were averaged to find the mean for visit one and the mean for visit two. These two values were then averaged. The difference between each instrument mean was then determined for each subject along with the standard deviation and 95% limits of agreement (the mean ± 1.96 x standard deviation). A Bland-Altman analysis was used to plot the mean and 95% limits of agreement for the Visante versus Orbscan, Visante versus ultrasound, and Orbscan versus

24 ultrasound for all cornea types together and for each separate cornea type. A two-sample t test was conducted for each of these comparisons to determine statistical significance.

For analysis of repeatability, three measurements taken by each instrument on each subject were averaged to find the mean for visit one and the mean for visit two. The difference between each visit mean was then determined for each subject along with the standard deviation and 95% limits of agreement. A Bland-Altman analysis was used to plot the mean and 95% limits of agreement for the Visante, Orbscan, and ultrasound for all cornea types together and for each separate cornea type. A paired two-sample t test was conducted for each of these comparisons to determine statistical significance.

CHAPTER 4

RESULTS

4.1 Overview

Raw pachymetry data are presented in Appendix B for all subjects and for both sessions. Three measurements were made with the Visante and the Orbscan II during each session. Orbscan data were unable to be collected on two subjects due to instrumentation malfunction. As suggested by the manufacturer, five measurements were made using the ultrasound pachymeter. The first three measurements were used for data analysis. In cases where any of the first three numbers appeared inconsistently higher or lower, the three most similar numbers were used for analysis.

4.2 Mean central corneal thickness

Table 1 shows the mean central corneal thickness of each technique and cornea type averaged across both sessions. Overall, ultrasound gave the highest mean central corneal thickness and, as expected normal corneas measured the thickest. For normal corneas, ultrasound measured the thickest mean central corneal thickness (±SD): 550 ±

34 μm; followed by the Orbscan II: 541 ± 44 μm; and the Visante: 532 ± 38 μm. For post-LASIK corneas, ultrasound gave the highest mean central corneal thickness: 502 ±

40 μm followed by the Orbscan II: 495 ± 52 μm and the Visante: 485 ± 42 μm. The

Orbscan and the Visante underestimate corneal thickness compared to ultrasound in normal and post-LASIK corneas, but Figure 4 shows that the differences among techniques are very similar for both LASIK and normal corneas. The results were slightly different for keratoconic corneas. Ultrasound again gave the thickest mean central corneal thickness (529 ± 46 μm), but the Visante (498 ± 44 μm) gave thicker measurements than the Orbscan (490 ± 53 μm).

Mixed model analysis of variance showed a significant interaction between instrument (p < 0.001) and cornea types (p = 0.012). In other words, the instruments not only measured significantly different from each other but this difference varied with cornea type. Because an interaction was found, Bland-Altman analysis and a paired two- sample t test were then conducted to determine which set of factors drove the interaction found in the mixed model analysis of variance (Figures 5 through 16).

When all three cornea types were analyzed as one group, the Visante measured on average 22 μm less than the ultrasound (Figure 5: 95% LoA = –55 to +11 μm; p < 0.001, t = −8.59); the Orbscan measured 18 μm less than the ultrasound (Figure 6: 95% LoA = –

68 to +32 μm; p = < 0.001, t = −4.64); and the Visante did not differ from the Orbscan

(Figure 7: 95% LoA = –41 to +31 μm; p = 0.093, t = −1.72). Thus, both the Visante and the Orbscan measured significantly different than the ultrasound, but the Visante did not measure significantly different from the Orbscan when all groups were analyzed together.

The three cornea types were then analyzed separately, and measurements made by each instrument were again compared.

For normal corneas, the Visante measured on average 18 μm less than the ultrasound (Figure 8: 95% LoA = –31 to –5 μm; p < 0.001, t = −10.44); the Orbscan measured 9 μm less than the ultrasound (Figure 9: 95% LoA = –34 to +16 μm; p = 0.016, t = −2.73); the Visante measured 9 μm less than the Orbscan (Figure 10: 95% LoA = –30 to +12 μm; p = 0.006, t = −3.24). Therefore in normal corneas, all three instruments measured significantly different from each other with the Visante versus ultrasound showing the greatest difference.

For post-LASIK corneas, the Visante measured on average 17 μm less than

ultrasound (Figure 11: 95% LoA = –31 to –2 μm; p < 0.001, t = −8.37); the Orbscan was

not significantly different from ultrasound (Figure 12: 95% LoA = –47 to +34 μm; p =

0.25, t = −1.21); and the Visante measured 10 μm less than the Orbscan (Figure 13: 95%

LoA = –38 to +18 μm; p = 0.014, t = −2.80). In post-LASIK corneas, the Visante is

measuring significantly different than ultrasound and the Orbscan but measuring a greater

difference compared to ultrasound.

For keratoconic corneas, the Visante measured on average 32 μm less than the

ultrasound (Figure 14: 95% LoA = –82 to +19 μm; p < 0.001, t = −4.73); the Orbscan

measured 41 μm less than the ultrasound (Figure 15: 95% LoA = –92 to +10 μm; p <

0.001, t = −5.72); and the Visante was not significantly different from the Orbscan

(Figure 16: 95% LoA = –45 to +56 μm; p = 0.44, t = 0.81). The Visante and the Orbscan

measured significantly different from ultrasound but not significantly different from each

other. Furthermore, for all comparisons the 95% of agreement span around 100 μm,

suggesting relatively poor agreement among the three techniques. Refer to tables 1 and 2

and figures 4 to 16.

Visante measures thinner than ultrasound regardless of subject group. Orbscan

measures thinner than ultrasound in normals and keratoconus but no different in LASIK.

Visante measures thinner than Orbscan normals and LASIK, but no different in

keratoconus (Table 2).

4.3 Between session repeatability

Bland-Altman analysis and a paired two-sample t test were conducted to assess

the repeatability of each technique. Table 3 summarizes the between-session

repeatability for all three techniques and all three groups. For each comparison the mean

difference between sessions is given along with the 95% LoA and the outcome of a paired t test. Overall, repeatability was poorest for ultrasound and for keratoconic corneas. When all three cornea types were analyzed as one group, the repeatability of the

Visante (Figure 17: 95% LoA = –21 to +23 μm; p = 0.67, t = –0.43) was similar to the

Orbscan (Figure 18: 95% LoA = –21 to +26 μm; p = 0.17, t = –1.42); and better than ultrasound (Figure 19: 95% LoA = –29 to +33 μm; p = 0.49, t = 0.71).

For normal corneas, the Visante was the most repeatable (Figure 20: 95% LoA =

–8 to +8 μm; p = 0.95, t = –0.06) followed by the Orbscan (Figure 21: 95% LoA = –11 to

+12 μm; p = 0.63, t = –0.48) and ultrasound (Figure 22: 95% LoA = –15 to +19 μm; p =

0.36, t = 0.95). All of these 95% LoA might be considered acceptable from a clinical perspective.

For post-LASIK corneas repeatability was very slightly poorer, with the Orbscan

(Figure 23: 95% LoA = –11 to +11 μm; p = 0.97; t = 0.05) and the Visante (Figure 24:

95% LoA = –10 to +13 µm; p = 0.27, t = –1.14) showing similar repeatability, and ultrasound (Figure 25: 95% LoA = –24 to +16 μm; p = 0.19, t = –1.38) being the least repeatable. Again, all of these 95% LoA might be considered acceptable from a clinical perspective.

For keratoconic corneas, the repeatability of the three instruments was very similar starting with the Visante (Figure 26: 95% LoA = –36 to +37 μm; p = 0.94, t = –

0.08); followed by the Orbscan (Figure 27: 95% LoA = –31 to +47 μm; p = 0.18, t = –

1.44); and ultrasound (Figure 28: 95% LoA = –40 to +53 μm; p = 0.30, t = 1.08).

Repeatability was poorest for ultrasound in keratoconic corneas but, in comparison to the other patient groups, the 95% LoA are relatively broad suggesting that repeatability is poor for all techniques.

It should be noted that the p and t values did not reach statistical significance for any of the above analyses. In other words, none of the instruments gave significantly different thickness measurements in the second session compared to the first session for any cornea type.

Visante Orbscan Ultrasound

Normal 532 ± 38 µm 541 ± 44 µm 550 ± 34 µm

Post-LASIK 485 ± 42 µm 495 ± 52 µm 502 ± 40 µm

Keratoconus 498 ± 44 µm 490 ± 53 µm 529 ± 46 µm

Table 1. Mean central corneal thickness for each group

Visante vs. Orbscan vs. Visante vs. Ultrasound Ultrasound Orbscan

Mean = −22 µm Mean = −18 µm Mean = −5 µm All LoA = −55 to +11µm LoA = −68 to +32 µm LoA = −41 to +31 µm (p < 0.001, t = −8.59) (p < 0.001, t = −4.64) (p = 0.093, t = −1.72)

Mean = −18 µm Mean = −9 µm Mean = −9 µm Normal LoA = −31 to −5 µm LoA = −34 to +16 µm LoA = −30 to +12 µm (p < 0.001, t −10.44) (p = 0.016, t = −2.73) (p = 0.006, t = −3.24)

Post- Mean = −17 µm Mean = −6 µm Mean = −10 µm LoA = −31 to −2 µm LoA = −47 to +34 µm LoA = −38 to +18 µm LASIK (p < 0.001, t = −8.37) (p = 0.25, t = −1.21) (p = 0.014, t = −2.80)

Mean = −32 µm Mean −41 µm Mean = +6 µm Keratoconus LoA = −82 to +19 µm LoA = −92 to +1 0 µm LoA = −45 to +56 µm (p < 0.001, t = −4.73) (p < 0.001, t = −5.72) (p = 0.44, t = +0.81)

Table 2. Validity for each instrument and cornea type. For each comparison, the mean, the 95% LoA are shown. The t-test results indicate whether or not a significant difference exists between the two instruments for the group of subjects

Visante Orbscan Ultrasound

Mean = +1 µm Mean = +2 µm Mean = +2 µm All LoA = −21 to +23 µm LoA = −21 to +26 µm LoA = −29 to +33 µm (p = 0.67, t = +0.43) (p = 0.165, t = +1.42) (p = 0.49, t = +0.71) Mean = 0 µm Mean = +1 µm Mean = +2 µm Normal LoA = −8 to +8 µm LoA = −11 to +12 µm LoA = −15 to +19 µm

(p = 0.95, t = +0.06) (p = 0.63, t −= +0.48) (p = 0.36, t = +0.95) Mean = +1 µm Mean = 0 µm Mean = −4 µm Post-LASIK LoA = −10 to +13 µm LoA = −11 to +11 µm LoA = −24 to +16 µm

(p = 0.27, t = +1.14) (p = 0.97, t = +0.05) (p = 0.19, t = −1.38) Mean = +1 µm Mean = +8 µm Mean = +6 µm Keratoconus LoA = −36 to +37 µm LoA = −31 to +47 µm LoA = −40 to +53 µm

(p = 0.94, t = +0.08) (p = 0.18, t +1.44) (p = 0.30, t = +1.08)

Table 3. Repeatability for each instrument and cornea type. For each comparison, the mean, the 95% LoA are shown. The t-test results indicate no significant difference exists between the two sessions for any instrument or group of subjects.

560 Visante Orbscan Ultrasound 540

520

500

480

460

440 Normal Post-LASIK Keratoconus Mean Corneal Thickness (um) Group

Figure 4. Results comparing mean central corneal thickness in each of the three subject groups and for each of the three techniques. Note the false zero on the y axis.

Figure 5. Validity: Visante versus Ultrasound for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 6. Validity: Orbscan versus Ultrasound for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 7. Validity: Visante versus Orbscan for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA. 35

Figure 8. Validity: Visante versus Ultrasound for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 9. Validity: Orbscan versus Ultrasound for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 10. Validity: Visante versus Orbscan for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA. 36

Figure 11. Validity: Visante versus Ultrasound for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 12. Validity: Orbscan versus Ultrasound for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 13. Validity: Visante versus Orbscan for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 14. Validity: Visante versus Ultrasound for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 15. Validity: Orbscan versus Ultrasound for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 16. Validity: Visante versus Orbscan for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 17. Repeatability of Visante for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 18. Repeatability of Orbscan for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 19. Repeatability of ultrasound for all cornea types. White dots are normal corneas, gray dots are keratoconic corneas, and black dots are post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA. 39

Figure 20. Repeatability of Visante for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 21. Repeatability of Orbscan for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 22. Repeatability of Ultrasound for normal corneas. The dashed lines show the mean difference (bias) and the 95% LoA. 40

Figure 23. Repeatability of Visante for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 24. Repeatability of Orbscan for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 25. Repeatability of Ultrasound for post-LASIK corneas. The dashed lines show the mean difference (bias) and the 95% LoA. 41

Figure 26. Repeatability of Visante for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 27. Repeatability of Orbscan for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

Figure 28. Repeatability of Ultrasound for keratoconic corneas. The dashed lines show the mean difference (bias) and the 95% LoA.

CHAPTER 5

DISCUSSION

5.1 Overview

The results of this study show that there was a significant interaction between the

different instruments and the types of corneas that were measured. When all cornea types were analyzed as one group, the Visante and Orbscan II gave statistically significant thinner values than ultrasound; the Visante did not measure significantly different from the Orbscan. The three cornea types were then analyzed separately. For normal corneas, the Visante measured on average 18 μm less than ultrasound; the Orbscan measured 9

μm less than the ultrasound; the Visante measured 9 μm less than the Orbscan. In normal corneas, all three instruments measured statistically significantly different from each other with the Visante versus ultrasound measuring the greatest difference.

For post-LASIK corneas, the Visante measured on average 17 μm less than ultrasound; the Orbscan no different from ultrasound; and the Visante measured 10 μm less than the Orbscan. In post-LASIK corneas, the Visante is measuring significantly different than ultrasound and the Orbscan but measuring a greater difference when compared to ultrasound. For keratoconic corneas, the Visante measured on average 32

μm less than the ultrasound; the Orbscan measured 41 μm less than the ultrasound; and

the Visante measured no different from the Orbscan. The Visante and the Orbscan

measured significantly different from ultrasound but not significantly different from each

other.

Repeatability was poorest for ultrasound and for keratoconic corneas. When all

three cornea types were analyzed as one group, the repeatability of the Visante was

similar to the Orbscan, and better than ultrasound. For normal corneas, the Visante was

the most repeatable and ultrasound the least repeatable. For post-LASIK corneas

repeatability was slightly poorer, with the Orbscan and the Visante showing similar

repeatability, and ultrasound being the least repeatable. For keratoconic corneas, the

repeatability of the three instruments was very similar starting with the Visante followed

by the Orbscan and ultrasound.

5.2 Central Corneal Thickness

The exact central corneal thickness (CCT) is not known, thus the literature serves

as a guideline for the accepted value of central corneal thickness. A meta-analysis by

Doughty reported an overall mean central corneal thickness of 536 ± 31μm for normal

corneas (Doughty and Zaman 2000). There have been variations reported in the literature

and many possible explanations have been proposed. In our study, ultrasound measured the thickest mean central corneal thickness in normal and post-LASIK corneas followed

by the Orbscan II and the Visante. Mean central corneal thickness in normal corneas was

very similar to that of Doughty’s meta- analysis (Doughty and Zaman 2000). For

keratoconic corneas, ultrasound measured the highest mean followed by the Visante and

the Orbscan II. Mixed model analysis showed a significant interaction between

instrument and cornea types. That is, the instruments not only measured significantly different from each other (p < 0.001) but also significantly different when measuring the various cornea types (p=0.012). See Table 3 through 5 for a comparison of our study findings to previous studies.

5.3 Repeatability

Repeatability is an important measure because it allows the clinician to make assessments of the state of the cornea over a period of time. For day-to-day comparison, all techniques showed acceptable repeatability when measuring normal and post-LASIK corneas. Repeatability was poorer for all techniques in the keratoconus corneas. For normal corneas, the Visante was the most repeatable followed by the Orbscan and ultrasound. Our findings are similar to several other studies that determined Orbscan to be more repeatable than ultrasound in normal corneas, see Table 6 for a comparison of our study findings to previous studies. For post-LASIK corneas, the Orbscan was the most repeatable but very similar to the Visante, and ultrasound was the least repeatable.

For keratoconic corneas, the Visante was the most repeatable followed by the Orbscan and ultrasound. The discrepancies in agreement and repeatability of each instrument may likely be attributable to the different methods of each technique. It is probable that the unique inherent properties of data acquisition may be affected differently in normal and non-physiologic corneal states. Very few studies have assessed repeatability in disease states. It is likely that the Visante and the Orbscan are able to more accurately detect the thinnest part of the cornea in keratoconus, whereas inaccurate probe position may lead to higher and less repeatable central corneal thickness measurements. Also, it is assumed

45 clinically, that keratoconic corneas do not fluctuate day-to-day. With poorer repeatability for every instrument however, it may be plausible that some fluctuations in the cornea do occur. To our knowledge, this is the first study to determine repeatability of these three instruments in corneas thinned by refractive surgery or by pathology.

5.4 Ultrasound Pachymetry

Though ultrasound has been considered the gold standard, its limitations have long been recognized including the need for topical anesthetic and subsequent contact with a probe. It only yields a single point measurement of which the accuracy and repeatability may be affected by inappropriate probe positioning and lack of gaze fixation

(Fishman, Pons et al. 2005). Because it requires corneal contact, it is difficult to accurately locate the same points of measurement in serial examinations and thus can affect repeatability (de Sanctis, Missolungi et al. 2007). In addition, Liu et al. suggested that contact with the cornea may affect measurements due to indentation of the cornea

(Liu, Huang et al. 1999). Solomon, however, demonstrated that corneal indentation does not affect ultrasound measures of corneal thickness (Solomon, Donnenfeld et al. 2004).

There have also been claims that ultrasound measurements may or may not be affected in corneal pathology or in post-refractive surgery cases. As the reference instrument, it is likely assumed that the ultrasound velocity is constant across all regions of the corneal tissue, though specific data on this do not appear to be available for human corneas (Doughty and Jonuscheit 2007). It has been the thought that ultrasound velocity decreases with increasing corneal edema subsequently increasing corneal thickness measurements in keratoconus corneas (Bechmann, Thiel et al. 2001; Haque, Simpson et

al. 2006). Conversely, Doughty et al. stated that no detectable time difference in

ultrasound transmission was demonstrated in animal corneas even when a 3-fold increase

in corneal hydration was artificially induced (Doughty and Jonuscheit 2007). In addition to corneal edema, it has been suggested that altered organization of collagen fibers, presence of anterior clear stromal spaces, uneven hydration of different parts of the stroma, and irregularities of the corneal endothelium or epithelium may modify the refractive index in keratoconus eyes (Ucakhan, Ozkan et al. 2006). These changes could not only shift the refractive index but also change the point of reflection, which is currently unknown. It is thought to be located between Descemet’s membrane and the anterior chamber (Zhao, Zou et al. 2007). It appears that corneal homogeneity may

influence both ultrasound and optical pachymetry measurements.

5.5 Orbscan Pachymetry

Many studies have demonstrated that the Orbscan II underestimates central

corneal thickness as compared to ultrasound in normal, keratoconus, and post-refractive

surgery subjects. In earlier publications, the Orbscan measured thicker than ultrasound,

and it had been suggested that the overestimation was due to the optical nature of the

Orbscan measuring the tear film. Conversely, it has been shown that the ultrasound probe

can displace 7 to 40 μm tear film (Nissen, Hjortdal et al. 1991). This explanation is

debatable due to differing tear film thicknesses that have been reported and is still an

unlikely cause of the magnitude of the discrepancy (Benedetto, Shah et al. 1975; Prydal

and Campbell 1992; Wang, Fonn et al. 2003; King-Smith, Fink et al. 2004). Haque et al.

conducted a study of corneal and epithelial thickness in subjects with keratoconus using

the Orbscan II and a Humphrey-Zeiss OCT system. They point out that if the tear film

was in fact ~40 μm thick, the OCT would have detected it as a separate layer. It was,

however, not detected and so strengthens the notion that the pre-corneal tear film is in

fact 3 to 7 μm thick and does not account for the magnitude of disagreement between

ultrasound and Orbscan measurements (Haque, Simpson et al. 2006).

In response to the discrepancy, the manufacturer of the Orbscan II included an acoustic factor that had been shown to produce closer agreement between the two

techniques, reducing the pachymetry measurement by about 8% compared to ultrasound

measurements (Cairns and McGhee 2005). Doughty analyzed 46 published studies of

6136 eye comparing average central corneal thickness measurements of ultrasound and

the Orbscan with and without the correction factor. He found that average central corneal

thickness without the correction factor was indeed higher (582 μm) than ultrasound (545

μm) and Orbscan with the correction factor (536 μm). He argues that overall, the level of

agreement between the Orbscan and ultrasound is limited (LoA without correction factor

0.004 to 0.073mm; LoA with correction factor –0.041 to +0.023 mm) and that Orbscan

data should be reported without any adjustment (Doughty and Jonuscheit 2010). In

addition, it has been proposed that the correction factor was calibrated in a normal

population, and thus provides disproportionately lower readings in post-LASIK corneas

(Kawana, Tokunaga et al. 2004). Fakhry et al. found that measurements of Orbscan II

with correction factor were comparable to ultrasound in normal corneas but not in

corneas with different degrees of corneal haze. There was a statistically significant

correlation between the grade of haze and the decrease in Orbscan measurements. They

48 suggested that corneal haze and/or stromal disorganization could affect corneal thickness measurements of optical techniques such as OCT and the Orbscan II (Fakhry, Artola et al. 2002). The Orbscan II assumes an index of refraction of 1.376 in generating pachymetry values. This could be a false assumption in corneas with altered collagen organization, anterior clear spaces, and uneven water distribution in the stroma due to the disease process of keratoconus (Gherghel, Hosking et al. 2004). Boscia et al. suggested that there is an increase in refractive index of hazy corneas due to the formation of new type III collagen and vacuoles filled with proteoglycan debris due to refractive surgery

(Boscia, La Tegola et al. 2002). Haque et al. also states that the presence of corneal haze in keratoconus can cause a change in refractive indexes and affect Orbscan measurements. Kawana et al. suggested that in corneas without clinically significant haze, the possibility exists that subtle, inhomogeneous changes could result in alteration of the corneal refractive indexes after refractive surgery (Kawana, Tokunaga et al. 2004;

Haque, Simpson et al. 2006).

In addition to possible changes in refractive indexes and stromal haze, Doughty points out that another source of potential error might lie in the customized and proprietary calculations made from the ray tracing of the 40 scans made across the cornea in the Orbscan. He states that presumably, assumptions and estimates are made relating to the curvature of the anterior and posterior surface with the anterior surface being the more likely one to be defined for the resultant topographic mapping of the corneal thickness profile (Doughty and Jonuscheit 2007). With the anterior surface being modified in refractive surgery, the reconstruction algorithms may not properly apply to

corneas with non-physiologic conditions such as post-LASIK eye (Kawana, Tokunaga et al. 2004). Avila et al., who found that the Orbscan significantly underestimated corneal thickness post-LASIK, also proposed the modified anterior topography as a possible source of error. According to the Orbscan II manual, the anterior and posterior boundaries are identified by the point of greatest gradient. Increased scattering from the

LASIK flap or interface may skew gradient measurement in Orbscan pachymetry (Avila,

Li et al. 2006). Maruoka et al. proposed that a change in the magnification ratio of the posterior cornea could help explain many of the discrepancies in the data obtained by the

Orbscan after keratorefractive surgery. They describe how the posterior surface of the cornea is observed through a “lens” composed of the overlying epithelium and stroma.

The “lens” changes its shape and thickness after LASIK, subsequently, the posterior surface of the cornea observed through the “lens” becomes relatively smaller. They state that this could be related to the apparent forward shift of the posterior cornea displayed with the Orbscan after myopic correction (Maruoka, Nawa et al. 2005).

5.6 Visante Pachymetry

As with the current study, Kim et al., Li et al., and Zhao et al. also found that the

Visante underestimated central corneal thickness as compared to ultrasound in normal

corneas (Li, Mohamed et al. 2007; Zhao, Wong et al. 2007; Kim, Budenz et al. 2008). It

is important to note that Kim et al. did not use the Visante but the Heidelberg anterior

segment OCT. Few studies have been conducted using the Visante; however, it is

plausible that the same considerations affecting Orbscan measurements could apply to

OCT due to its optical nature. An extra consideration is the uncertainty of the true index

50 of refraction of infrared radiation in the cornea, which could create a source of error in central corneal thickness calculations (Kim, Budenz et al. 2008). Li et al. found that the

Visante underestimated central corneal thickness relative to ultrasound but underestimated to a lesser degree in normal, thinner corneas, suggesting better agreement between the Visante and ultrasound in thinner corneas. The Orbscan II overestimated in thinner corneas, thus they concluded that the Visante may be a suitable and better alternative to the Orbscan II for corneal thickness measurements (Li, Mohamed et al.

2007). Our present study found that the Orbscan II measured thicker than the Visante in normal and post-Lasik corneas but thinner than the Visante in keratoconus corneas. The

Visante uses scanning infrared spots that move across the corneal surface with multiple axial scans consisting of up to 2,048 A-scans. This may allow for better delineation of the corneal surfaces with the tracing algorithm software, and the more rapid acquisition time may also reduce errors due to patient movement. More studies are needed to assess the

Visante’s role in clinical and research settings.

5.7 Summary

In deciding which instrument the clinician will use to measure central corneal thickness, not only must the pros and cons of each instrument must be considered but also the bias that may exist when normal and thinned corneas are measured. Ultrasound pachymetry has long been the gold standard and is portable and less costly compared to the optical instruments. The optical techniques, however, provide more information about the entire cornea and do not require contact. The data acquired by these techniques can be invaluable to clinician. Because of the visualization capabilities of the Visante and

51 topographic capabilities of the Orbscan II, clinicians may be able to better detect sub- clinical keratoconus, follow the progression of disease, and assess pre- and post- keratorefractive and keratoplasty outcomes. Though repeatability was poorer for all techniques in the keratoconus corneas and the measurements significantly differed between each instrument, a case must still be made for the importance of the Visante and

Orbscan II. It is important to note that in regards to pachymetry, CCT acquired by the

Visante and Orbscan are not yet considered interchangeable with ultrasound.

5.8 Limitations

While this study was successful in determining the repeatability and validity of these three instruments in normal and thinned corneas, there are some limitations to the study that need to be addressed. First, CCT measurements should have been made at the same time of day for each subject. While a concerted effort was made to do this, it was not always successful. Second, the subjects should have been provided with a better, more sophisticated fixation target when taking ultrasound measurements because it can be difficult to estimate the center of the cornea on every occasion. Third, while every effort was made to assure that only one examiner was taking the measurements, there were a few occasions where this was not possible. It could be argued that the outcome might be slightly affected due to a more skilled clinician taking a few of the measurements. Fourth, keratoconus subjects were not included or excluded based on the severity of the disease. Further studies may be needed to assess the validity and repeatability in certain stages of disease. Fifth, Orbscan data could not be collected on two subjects due to instrument technical problems.

Orbscan µm Ultrasound Orbscan Author Correction Visante µm µm µm Factor Doughty 2007 525 ± 35 579 ± 37 Fishman et al. 2005 545 ± 38 574 ± 51* Fernandez et al. 2002 551 ± 5 560 ± 4 Gonzalez-Meijome et 545 ± 40 560 ± 48 al. 2003 Marsich 2000 542 ± 33 596 ± 40* Hashemi et al. 2007 535 ± 28 557 ± 31 Doughty 2007 523 ± 37 591 ± 44 Yaylali et al. 1997 543 ± 8 571 ± 6 Gherghel et al. 2004 551 ± 37 594 ± 52 546 ± 48 Lackner et al. 2005 552 ± 32 576 ± 37 530 ± 34 Hashemi 2007 555 ± 30 580 ± 40 533 ± 37 Amano et al. 2005 545 ± 31 541 ±41 Radford et al. 2004 558 ± 6 568 ± 6 Suzuki et al. 2003 548 ±33 547 ±35 Rainer et al. 2004 545 ± 35 543 ± 35* McLaren et al. 2004 555 ± 28 540 ± 35 Haque et al. 2005 531 ± 30 513 ± 44 Fakhry et al. 2002 528 ± 54 530 ± 55 Yazici et al. 2010 554 ± 33 529 ± 31 Mohamed 2007 543 ± 34 Zhao et al. 2007 542 ± 37 527 ± 34 Li et al. 2007 554 ± 30 553 ± 26† 539 ± 26 Present Study 550 ± 34 541 ± 44 532 ± 38

Table 4. Studies comparing normal corneas * Orbscan I † Used a 0.89 correction factor

Orbscan µm Orbscan Author Ultrasound µm Correction Visante µm µm Factor Hashemi 2007 478 ± 51 474 ± 67 436 ± 62 Fakhry et al. 478 ± 87 510 ± 72 2002 Iskander et al. 493 ± 42 431 ± 42 2001 Kawana et al. 479 ± 42 446 ± 60 2004 Ho et al. 2007 438 ± 41 435 ± 50† 427 ± 42 Cheng et al. 437 ± 44 423 ± 51† 422 ± 43 2008 Present Study 502 ± 40 495 ± 52 485 ± 42

Table 5. Studies comparing post-LASIK corneas * Orbscan I † Used a 0.89 correction factor

Orbscan µm Orbscan Author Ultrasound µm Correction Visante µm µm Factor Haque et al. 494 ± 50 439 ± 48 2005 Gherghel et al. 503 ± 53 499 ± 68 459 ± 63 2004 Mohamed 2007 467.3 ± 49.3 Kawana et al. 485 ± 29 450 ± 43 2005 Present Study 529 ± 46 490 ± 53 498 ± 44

Table 6. Studies comparing keratoconic corneas

Author Ultrasound Orbscan Visante

Lackner et al. 95 % LoA 95 % LoA

2005 – 6.7, +7.3 –12, +14 95 % LoA 95% LoA Marsich 2000 –22 to +24 –10 to +17 Bourges et al ICCs ICCs

2009 0.608–0.958 0.967–0.992 Variation mean Variation mean Amano et al. ± SD ± SD

2006 1.6 ± 0.7 4.5 ± 3.2 0.30 ± 0.13% 0.84 ± 0.60% Variation mean Variation mean Suzuki et al. ± SD ± SD

2003 4.61 ± 2.33 4.88 ± 2.91 0.86 ± 0.44% 0.89 ± 0.54% 95% LoA 95% LoA 95% LoA Present Study −29 to +33 −21 to +26 −21 to +23

Table 7. Studies comparing repeatability in normal corneas

CHAPTER 6

CONCLUSION

Corneal thickness is a valuable indicator of the health and physiology of the

cornea. Central corneal thickness measurement is not only vitally important for the

diagnosis, treatment, and management of various ocular conditions but also a crucial

parameter in determining the safety of contact lens wear and refractive surgery.

Ultrasound pachymetry has long been considered the gold standard, however, there has been the advent of more sophisticated and versatile instruments. This study compared the validity and repeatability of the Orbscan II topographer and the Visante anterior segment

optical coherence tomographer to ultrasound in normal and thinned corneas. From this

study we conclude that:

1. The between session repeatability of the instruments when measuring normal corneas

can be ranked as follows: Visante>Orbscan II>ultrasound.

2. The between session repeatability of the instruments when measuring post-LASIK

corneas can be ranked as follows: Orbscan II>Visante>ultrasound.

3. The between session repeatability of the instruments when measuring keratoconus

corneas can be ranked as follows: Visante>Orbscan II>ultrasound.

4. The between session repeatability was poorer for all techniques when measuring

keratoconus corneas versus normal or post-LASIK corneas.

5. A significant interaction was found between instrument and cornea type, thus the

instruments not only measured significantly different from each other (p < 0.001) but also

significantly different when measuring the various cornea types (p = 0.012).

6. Mean central corneal thickness in normal corneas was measured by each instrument as

follows: ultrasound > Orbscan II > Visante; all three instruments measured statistically significantly different from each other.

7. Mean central corneal thickness in post-LASIK corneas was measured by each instrument as follows: ultrasound = Orbscan II > Visante; the Visante is measured

significantly different than ultrasound and the Orbscan but measuring a greater difference

when compared to ultrasound.

8. Mean central corneal thickness in keratoconus corneas was measured by each

instrument as follows: ultrasound > Visante = Orbscan II; Visante and the Orbscan

measured significantly different from ultrasound but not significantly different from each

other.

Appendix

Tables of individual subject data

Age in years 23 Gender Male Race African American Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -0.75 Cylinder 0.00 Axis 0 K's K 41.87 Axis 145 K 42.55 Axis 55 Visante 1st Visit 1 508 2 512 3 517 Mean 512 Visante 2nd Visit 1 508 2 513 3 516 Mean 512 Orbscan 1st Visit 1 527 2 521 3 529 Mean 526 Orbscan 2nd Visit 1 526 2 533 3 523 Mean 527 Ultrasound 1st Visit 1 549 2 539 3 534 Mean 541 Ultrasound 2nd Visit 1 526 2 520 3 529 Mean 525

Table 8: Demographics and raw pachymetry in μm for subject 1

Age in years 31 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 6 yrs Rx Sphere -1.25 Cylinder 0.00 Axis 0 K's K 40.62 Axis 4 K 41.75 Axis 94 Visante 1st Visit 1 482 2 481 3 481 Mean 481 Visante 2nd Visit 1 479 2 477 3 477 Mean 478 Orbscan 1st Visit 1 494 2 493 3 494 Mean 494 Orbscan 2nd Visit 1 489 2 496 3 493 Mean 493 Ultrasound 1st Visit 1 490 2 486 3 487 Mean 488 Ultrasound 2nd Visit 1 486 2 487 3 484 Mean 486

Table 9: Demographics and raw pachymetry in μm for subject 2

Age in years 23 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -1.50 Cylinder -0.62 Axis 96 K's K 40.87 Axis 17 K 40.5 Axis 107 Visante 1st Visit 1 551 2 553 3 553 Mean 552 Visante 2nd Visit 1 555 2 558 3 552 Mean 555 Orbscan 1st Visit 1 559 2 550 3 557 Mean 555 Orbscan 2nd Visit 1 554 2 555 3 552 Mean 554 Ultrasound 1st Visit 1 572 2 565 3 567 Mean 568 Ultrasound 2nd Visit 1 573 2 568 3 562 Mean 568

Table 10: Demographics and raw pachymetry in μm for subject 3

Age in years 27 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -1.37 Cylinder -0.25 Axis 96 K's K 43.25 Axis 160 K 43.62 Axis 70 Visante 1st Visit 1 501 2 499 3 500 Mean 500 Visante 2nd Visit 1 494 2 491 3 497 Mean 494 Orbscan 1st Visit 1 500 2 494 3 496 Mean 497 Orbscan 2nd Visit 1 491 2 493 3 493 Mean 492 Ultrasound 1st Visit 1 512 2 510 3 505 Mean 509 Ultrasound 2nd Visit 1 509 2 512 3 513 Mean 511

Table 11: Demographics and raw pachymetry in μm for subject 4

Age in years 23 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -1.00 Cylinder 0.00 Axis 0 K's K 43.62 Axis 172 K 45.12 Axis 82 Visante 1st Visit 1 517 2 502 3 510 Mean 510 Visante 2nd Visit 1 510 2 511 3 503 Mean 508 Orbscan 1st Visit 1 528 2 525 3 523 Mean 525 Orbscan 2nd Visit 1 521 2 524 3 527 Mean 524 Ultrasound 1st Visit 1 529 2 548 3 528 Mean 535 Ultrasound 2nd Visit 1 520 2 515 3 518 Mean 518

Table 12: Demographics and raw pachymetry in μm for subject 5

Age in years 22 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -4.75 Cylinder -0.75 Axis 6 K's K 43 Axis 7 K 44.62 Axis 97 Visante 1st Visit 1 531 2 538 3 539 Mean 536 Visante 2nd Visit 1 527 2 530 3 533 Mean 530 Orbscan 1st Visit 1 548 2 547 3 544 Mean 546 Orbscan 2nd Visit 1 549 2 546 3 541 Mean 545 Ultrasound 1st Visit 1 549 2 547 3 549 Mean 548 Ultrasound 2nd Visit 1 544 2 544 3 543 Mean 544

Table 13: Demographics and raw pachymetry in μm for subject 6

Age in years 23 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -6.12 Cylinder -0.25 Axis 6 K's K 43.12 Axis 5 K 43.87 Axis 95 Visante 1st Visit 1 512 2 514 3 517 Mean 514 Visante 2nd Visit 1 512 2 511 3 511 Mean 511 Orbscan 1st Visit 1 523 2 518 3 516 Mean 519 Orbscan 2nd Visit 1 525 2 522 3 515 Mean 521 Ultrasound 1st Visit 1 539 2 530 3 532 Mean 534 Ultrasound 2nd Visit 1 527 2 527 3 531 Mean 528

Table 14: Demographics and raw pachymetry in μm for subject 7

Age in years 60 Gender Female Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 31 yrs Rx Sphere -14.12 Cylinder -2.75 Axis 58 K's K 60.25 Axis 167 K 62 Axis 77 Visante 1st Visit 1 430 2 422 3 422 Mean 425 Visante 2nd Visit 1 400 2 405 3 399 Mean 401 Orbscan 1st Visit 1 423 2 445 3 435 Mean 434 Orbscan 2nd Visit 1 440 2 439 3 446 Mean 442 Ultrasound 1st Visit 1 468 2 465 3 461 Mean 465 Ultrasound 2nd Visit 1 492 2 489 3 478 Mean 486

Table 15: Demographics and raw pachymetry in μm for subject 8

Age in years 22 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -9.87 Cylinder -0.62 Axis 121 K's K 44.87 Axis 20 K 44.37 Axis 110 Visante 1st Visit 1 541 2 542 3 547 Mean 543 Visante 2nd Visit 1 545 2 544 3 530 Mean 540 Orbscan 1st Visit 1 570 2 580 3 583 Mean 578 Orbscan 2nd Visit 1 566 2 571 3 577 Mean 571 Ultrasound 1st Visit 1 566 2 563 3 571 Mean 567 Ultrasound 2nd Visit 1 565 2 569 3 560 Mean 565

Table 16: Demographics and raw pachymetry in μm for subject 9

Age in years 31 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -5.50 Cylinder -0.37 Axis 17 K's K 44.37 Axis 2 K 45 Axis 92 Visante 1st Visit 1 524 2 529 3 524 Mean 526 Visante 2nd Visit 1 530 2 529 3 526 Mean 528 Orbscan 1st Visit 1 545 2 538 3 543 Mean 542 Orbscan 2nd Visit 1 541 2 537 3 544 Mean 541 Ultrasound 1st Visit 1 557 2 551 3 565 Mean 558 Ultrasound 2nd Visit 1 550 2 544 3 547 Mean 547

Table 17: Demographics and raw pachymetry in μm for subject 10

Age in years 44 Gender Male Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 9 yrs Rx Sphere -0.87 Cylinder -0.25 Axis 112 K's K 41.75 Axis 164 K 42 Axis 74 Visante 1st Visit 1 551 2 552 3 548 Mean 550 Visante 2nd Visit 1 551 2 551 3 554 Mean 552 Orbscan 1st Visit 1 561 2 562 3 565 Mean 563 Orbscan 2nd Visit 1 563 2 567 3 568 Mean 566 Ultrasound 1st Visit 1 571 2 581 3 578 Mean 577 Ultrasound 2nd Visit 1 565 2 561 3 563 Mean 563

Table 18: Demographics and raw pachymetry in μm for subject 11

Age in years 56 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -11.50 Cylinder -0.87 Axis 20 K's K 44.75 Axis 6 K 45.87 Axis 96 Visante 1st Visit 1 552 2 555 3 562 Mean 556 Visante 2nd Visit 1 559 2 561 3 562 Mean 561 Orbscan 1st Visit 1 559 2 562 3 560 Mean 560 Orbscan 2nd Visit 1 570 2 571 3 567 Mean 569 Ultrasound 1st Visit 1 580 2 574 3 575 Mean 576 Ultrasound 2nd Visit 1 573 2 574 3 573 Mean 573

Table 19: Demographics and raw pachymetry in μm for subject 12

Age in years 27 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -4.50 Cylinder -0.25 Axis 15 K's K 43 Axis 13 K 43.62 Axis 103 Visante 1st Visit 1 535 2 535 3 535 Mean 535 Visante 2nd Visit 1 536 2 536 3 536 Mean 536 Orbscan 1st Visit 1 542 2 544 3 539 Mean 542 Orbscan 2nd Visit 1 552 2 552 3 552 Mean 552 Ultrasound 1st Visit 1 561 2 576 3 554 Mean 564 Ultrasound 2nd Visit 1 563 2 572 3 557 Mean 564

Table 20: Demographics and raw pachymetry in μm for subject 13

Age in years 24 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 2 yrs Rx Sphere -1.50 Cylinder -0.25 Axis 169 K's K 38.25 Axis 2 K 40.12 Axis 92 Visante 1st Visit 1 435 2 424 3 427 Mean 429 Visante 2nd Visit 1 424 2 436 3 436 Mean 432 Orbscan 1st Visit 1 438 2 432 3 430 Mean 433 Orbscan 2nd Visit 1 430 2 444 3 430 Mean 435 Ultrasound 1st Visit 1 457 2 469 3 456 Mean 461 Ultrasound 2nd Visit 1 448 2 447 3 453 Mean 449

Table 21: Demographics and raw pachymetry in μm for subject 14

Age in years 25 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -4.62 Cylinder -0.50 Axis 20 K's K 42.87 Axis 5 K 44 Axis 95 Visante 1st Visit 1 545 2 550 3 550 Mean 548 Visante 2nd Visit 1 555 2 549 3 551 Mean 552 Orbscan 1st Visit 1 562 2 563 3 564 Mean 563 Orbscan 2nd Visit 1 563 2 565 3 557 Mean 562 Ultrasound 1st Visit 1 558 2 558 3 560 Mean 559 Ultrasound 2nd Visit 1 567 2 561 3 566 Mean 565

Table 22: Demographics and raw pachymetry in μm for subject 15

Age in years 50 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -2.37 Cylinder 0.00 Axis 0 K's K 43.37 Axis 9 K 45.12 Axis 99 Visante 1st Visit 1 580 2 578 3 572 Mean 577 Visante 2nd Visit 1 570 2 573 3 572 Mean 572 Orbscan 1st Visit 1 580 2 582 3 575 Mean 579 Orbscan 2nd Visit 1 575 2 582 3 584 Mean 580 Ultrasound 1st Visit 1 579 2 582 3 583 Mean 581 Ultrasound 2nd Visit 1 587 2 581 3 582 Mean 583

Table 23: Demographics and raw pachymetry in μm for subject 16

Age in years 51 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere 0.37 Cylinder -1.12 Axis 107 K's K 43.5 Axis 155 K 44.12 Axis 65 Visante 1st Visit 1 544 2 545 3 545 Mean 545 Visante 2nd Visit 1 546 2 546 3 549 Mean 547 Orbscan 1st Visit 1 550 2 552 3 562 Mean 555 Orbscan 2nd Visit 1 554 2 544 3 545 Mean 548 Ultrasound 1st Visit 1 554 2 569 3 565 Mean 563 Ultrasound 2nd Visit 1 567 2 564 3 563 Mean 565

Table 24: Demographics and raw pachymetry in μm for subject 17

Age in years 49 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 7 yrs Rx Sphere 0.25 Cylinder -1.00 Axis 86 K's K 40.5 Axis 35 K 41 Axis 125 Visante 1st Visit 1 517 2 517 3 519 Mean 518 Visante 2nd Visit 1 518 2 518 3 515 Mean 517 Orbscan 1st Visit 1 525 2 528 3 528 Mean 527 Orbscan 2nd Visit 1 527 2 522 3 532 Mean 527 Ultrasound 1st Visit 1 537 2 536 3 535 Mean 536 Ultrasound 2nd Visit 1 532 2 537 3 534 Mean 534

Table 25: Demographics and raw pachymetry in μm for subject 18

Age in years 59 Gender Female Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 32 yrs Rx Sphere -2.87 Cylinder -1.62 Axis 53 K's K 45.37 Axis 35 K 47.75 Axis 125 Visante 1st Visit 1 458 2 455 3 448 Mean 454 Visante 2nd Visit 1 483 2 479 3 480 Mean 481 Orbscan 1st Visit 1 460 2 468 3 474 Mean 467 Orbscan 2nd Visit 1 483 2 465 3 495 Mean 481 Ultrasound 1st Visit 1 509 2 500 3 495 Mean 501 Ultrasound 2nd Visit 1 505 2 524 3 516 Mean 515

Table 26: Demographics and raw pachymetry in μm for subject 19

Age in years 22 Gender Male Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere -2.62 Cylinder -0.87 Axis 4 K's K 44.25 Axis 10 K 45 Axis 100 Visante 1st Visit 1 431 2 432 3 423 Mean 429 Visante 2nd Visit 1 445 2 436 3 431 Mean 437 Orbscan 1st Visit 1 411 2 410 3 410 Mean 410 Orbscan 2nd Visit 1 421 2 429 3 420 Mean 423 Ultrasound 1st Visit 1 457 2 458 3 446 Mean 454 Ultrasound 2nd Visit 1 474 2 469 3 470 Mean 471

Table 27. Demographics and raw pachymetry in μm for subject 20

Age in years 34 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 3 yrs Rx Sphere 0.00 Cylinder -0.25 Axis 30 K's K 44.37 Axis 2 K 45.25 Axis 92 Visante 1st Visit 1 469 2 467 3 470 Mean 469 Visante 2nd Visit 1 477 2 486 3 480 Mean 481 Orbscan 1st Visit 1 522 2 515 3 517 Mean 518 Orbscan 2nd Visit 1 501 2 514 3 502 Mean 506 Ultrasound 1st Visit 1 478 2 483 3 477 Mean 479 Ultrasound 2nd Visit 1 477 2 486 3 480 Mean 481

Table 28: Demographics and raw pachymetry in μm for subject 21

Age in years 56 Gender Female Race Caucasian Cornea type Normal Duration after Lasik or with Keratoconus NA Rx Sphere 1.12 Cylinder 0.00 Axis 0 K's K 43.25 Axis 165 K 44.5 Axis 75 Visante 1st Visit 1 600 2 597 3 593 Mean 597 Visante 2nd Visit 1 603 2 596 3 595 Mean 598 Orbscan 1st Visit 1 608 2 601 3 617 Mean 609 Orbscan 2nd Visit 1 607 2 609 3 609 Mean 608 Ultrasound 1st Visit 1 614 2 604 3 601 Mean 606 Ultrasound 2nd Visit 1 604 2 605 3 606 Mean 605

Table 29: Demographics and raw pachymetry in μm for subject 22

Age in years 53 Gender Male Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 6 yrs Rx Sphere -0.62 Cylinder -0.25 Axis 158 K's K 40.25 Axis 167 K 41.5 Axis 77 Visante 1st Visit 1 499 2 498 3 499 Mean 499 Visante 2nd Visit 1 499 2 497 3 507 Mean 501 Orbscan 1st Visit 1 531 2 530 3 524 Mean 528 Orbscan 2nd Visit 1 522 2 518 3 515 Mean 518 Ultrasound 1st Visit 1 509 2 511 3 516 Mean 512 Ultrasound 2nd Visit 1 506 2 506 3 508 Mean 507

Table 30: Demographics and raw pachymetry in μm for subject 23

Age in years 50 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 9 yrs Rx Sphere -0.50 Cylinder -0.37 Axis 60 K's K 41.37 Axis 5 K 42.12 Axis 95 Visante 1st Visit 1 558 2 558 3 557 Mean 558 Visante 2nd Visit 1 559 2 559 3 560 Mean 559 Orbscan 1st Visit 1 574 2 569 3 574 Mean 572 Orbscan 2nd Visit 1 569 2 580 3 577 Mean 575 Ultrasound 1st Visit 1 563 2 565 3 571 Mean 566 Ultrasound 2nd Visit 1 579 2 565 3 574 Mean 573

Table 31: Demographics and raw pachymetry in μm for subject 24

Age in years 44 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 10 yrs Rx Sphere -1.50 Cylinder 0.00 Axis 0 K's K 44.5 Axis 26 K 45.37 Axis 116 Visante 1st Visit 1 527 2 526 3 523 Mean 525 Visante 2nd Visit 1 520 2 522 3 513 Mean 518 Orbscan 1st Visit 1 542 2 536 3 533 Mean 537 Orbscan 2nd Visit 1 541 2 539 3 521 Mean 534 Ultrasound 1st Visit 1 539 2 540 3 549 Mean 543 Ultrasound 2nd Visit 1 543 2 542 3 545 Mean 543

Table 32: Demographics and raw pachymetry in μm for subject 25

Age in years 40 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 8 yrs Rx Sphere 0.00 Cylinder -0.50 Axis 5 K's K 42 Axis 137 K 42.12 Axis 47 Visante 1st Visit 1 471 2 471 3 470 Mean 471 Visante 2nd Visit 1 476 2 476 3 473 Mean 475 Orbscan 1st Visit 1 470 2 466 3 474 Mean 470 Orbscan 2nd Visit 1 475 2 471 3 479 Mean 475 Ultrasound 1st Visit 1 487 2 489 3 482 Mean 486 Ultrasound 2nd Visit 1 522 2 516 3 484 Mean 507

Table 33: Demographics and raw pachymetry in μm for subject 26

Age in years 21 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 1 yr Rx Sphere -0.87 Cylinder 0.00 Axis 0 K's K 37.75 Axis 166 K 38.62 Axis 76 Visante 1st Visit 1 479 2 479 3 490 Mean 483 Visante 2nd Visit 1 477 2 474 3 480 Mean 477 Orbscan 1st Visit 1 496 2 496 3 499 Mean 497 Orbscan 2nd Visit 1 508 2 491 3 499 Mean 499 Ultrasound 1st Visit 1 491 2 486 3 489 Mean 489 Ultrasound 2nd Visit 1 491 2 497 3 500 Mean 496

Table 34: Demographics and raw pachymetry in μm for subject 27

Age in years 25 Gender Male Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 4 yrs Rx Sphere -1.00 Cylinder -0.50 Axis 151 K's K 40.75 Axis 158 K 42.37 Axis 88 Visante 1st Visit 1 482 2 481 3 481 Mean 481 Visante 2nd Visit 1 482 2 481 3 485 Mean 483 Orbscan 1st Visit 1 484 2 490 3 491 Mean 488 Orbscan 2nd Visit 1 484 2 490 3 491 Mean 488 Ultrasound 1st Visit 1 519 2 494 3 497 Mean 503 Ultrasound 2nd Visit 1 519 2 494 3 497 Mean 503

Table 35: Demographics and raw pachymetry in μm for subject 28

Age in years 63 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 5 yrs Rx Sphere -0.87 Cylinder -0.87 Axis 20 K's K 38.75 Axis 11 K 40.37 Axis 101 Visante 1st Visit 1 479 2 478 3 489 Mean 482 Visante 2nd Visit 1 497 2 498 3 492 Mean 496 Orbscan 1st Visit 1 488 2 505 3 486 Mean 493 Orbscan 2nd Visit 1 505 2 496 3 502 Mean 501 Ultrasound 1st Visit 1 497 2 488 3 492 Mean 492 Ultrasound 2nd Visit 1 508 2 503 3 514 Mean 508

Table 36: Demographics and raw pachymetry in μm for subject 29

Age in years 30 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 1 yr Rx Sphere 0.25 Cylinder 0.00 Axis 0 K's K 38 Axis 1 K 40 Axis 91 Visante 1st Visit 1 392 2 397 3 392 Mean 394 Visante 2nd Visit 1 392 2 391 3 390 Mean 391 Orbscan 1st Visit 1 357 2 358 3 368 Mean 361 Orbscan 2nd Visit 1 366 2 365 3 361 Mean 364 Ultrasound 1st Visit 1 406 2 426 3 423 Mean 418 Ultrasound 2nd Visit 1 430 2 428 3 422 Mean 427

Table 37: Demographics and raw pachymetry in μm for subject 30

Age in years 45 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 7 yrs Rx Sphere -0.75 Cylinder 0.00 Axis 0 K's K 42.87 Axis 167 K 43.5 Axis 77 Visante 1st Visit 1 464 2 462 3 444 Mean 457 Visante 2nd Visit 1 463 2 468 3 462 Mean 464 Orbscan 1st Visit 1 460 2 469 3 462 Mean 464 Orbscan 2nd Visit 1 476 2 469 3 466 Mean 470 Ultrasound 1st Visit 1 478 2 472 3 474 Mean 475 Ultrasound 2nd Visit 1 497 2 485 3 499 Mean 494

Table 38: Demographics and raw pachymetry in μm for subject 31

Age in years 53 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 22 yrs Rx Sphere -5.00 Cylinder -4.75 Axis 79 K's K 48.75 Axis 150 K 45.62 Axis 60 Visante 1st Visit 1 525 2 513 3 506 Mean 515 Visante 2nd Visit 1 548 2 556 3 546 Mean 550 Orbscan 1st Visit 1 542 2 542 3 543 Mean 542 Orbscan 2nd Visit 1 568 2 568 3 575 Mean 570 Ultrasound 1st Visit 1 618 2 619 3 618 Mean 618 Ultrasound 2nd Visit 1 628 2 624 3 626 Mean 626

Table 39: Demographics and raw pachymetry in μm for subject 32

Age in years 23 Gender Female Race Caucasian Cornea type LASIK Duration after Lasik or with Keratoconus 2 yrs Rx Sphere -1.12 Cylinder -0.37 Axis 13 K's K 38.5 Axis 153 K 39.75 Axis 63 Visante 1st Visit 1 462 2 475 3 465 Mean 467 Visante 2nd Visit 1 475 2 455 3 468 Mean 466 Orbscan 1st Visit 1 488 2 488 3 475 Mean 484 Orbscan 2nd Visit 1 475 2 471 3 486 Mean 477 Ultrasound 1st Visit 1 477 2 471 3 470 Mean 473 Ultrasound 2nd Visit 1 468 2 487 3 492 Mean 482

Table 40: Demographics and raw pachymetry in μm for subject 33

Age in years 39 Gender Male Race African American Cornea type Keratoconus Duration after Lasik or with Keratoconus 1 yr Rx Sphere 0.25 Cylinder -4.50 Axis 67 K's K 43.62 Axis 36 K 54.12 Axis 126 Visante 1st Visit 1 457 2 435 3 439 Mean 444 Visante 2nd Visit 1 460 2 463 3 456 Mean 460 Orbscan 1st Visit 1 446 2 463 3 425 Mean 445 Orbscan 2nd Visit 1 457 2 439 3 450 Mean 449 Ultrasound 1st Visit 1 476 2 507 3 480 Mean 488 Ultrasound 2nd Visit 1 495 2 488 3 485 Mean 489

Table 41: Demographics and raw pachymetry in μm for subject 34 (Subject 35 was lost to follow-up).

Age in years 72 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 50 yrs Rx Sphere -15.00 Cylinder -4.75 Axis 180 K's K 56 Axis 10 K 52 Axis 100 Visante 1st Visit 1 486 2 476 3 491 Mean 484 Visante 2nd Visit 1 465 2 433 3 428 Mean 442 Orbscan 1st Visit 1 469 2 412 3 401 Mean 427 Orbscan 2nd Visit 1 492 2 484 3 489 Mean 488 Ultrasound 1st Visit 1 517 2 488 3 477 Mean 494 Ultrasound 2nd Visit 1 490 2 506 3 468 Mean 488

Table 42: Demographics and raw pachymetry in μm for subject 36

Age in years 45 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 13 yrs Rx Sphere -7.00 Cylinder -1.12 Axis 32 K's K 42.87 Axis 37 K 44.12 Axis 127 Visante 1st Visit 1 496 2 498 3 496 Mean 497 Visante 2nd Visit 1 490 2 489 3 481 Mean 487 Orbscan 1st Visit 1 2 3 Mean Orbscan 2nd Visit 1 2 3 Mean Ultrasound 1st Visit 1 508 2 503 3 504 Mean 505 Ultrasound 2nd Visit 1 504 2 501 3 503 Mean 503

Table 43: Demographics and raw pachymetry in μm for subject 37

Age in years 49 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 20 yrs Rx Sphere -3.25 Cylinder -3.50 Axis 74 K's K 43 Axis 33 K 51.5 Axis 123 Visante 1st Visit 1 528 2 520 3 529 Mean 526 Visante 2nd Visit 1 528 2 524 3 526 Mean 526 Orbscan 1st Visit 1 2 3 Mean Orbscan 2nd Visit 1 519 2 523 3 513 Mean 518 Ultrasound 1st Visit 1 558 2 554 3 544 Mean 552 Ultrasound 2nd Visit 1 526 2 529 3 513 Mean 523

Table 44: Demographics and raw pachymetry in μm for subject 38

Age in years 62 Gender Female Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 35 yrs Rx Sphere -11.25 Cylinder -1.37 Axis 43 K's K 45.87 Axis 26 K 49.62 Axis 116 Visante 1st Visit 1 516 2 502 3 505 Mean 508 Visante 2nd Visit 1 518 2 503 3 495 Mean 505 Orbscan 1st Visit 1 2 3 Mean Orbscan 2nd Visit 1 2 3 Mean Ultrasound 1st Visit 1 547 2 524 3 552 Mean 541 Ultrasound 2nd Visit 1 473 2 476 3 471 Mean 473

Table 45: Demographics and raw pachymetry in μm for subject 39 (Subject 40 was lost to follow-up).

Age in years 50 Gender Female Race African American Cornea type Keratoconus Duration after Lasik or with Keratoconus 28 yrs Rx Sphere -12.37 Cylinder -4.62 Axis 172 K's K 44.62 Axis 150 K 49 Axis 60 Visante 1st Visit 1 601 2 600 3 595 Mean 599 Visante 2nd Visit 1 586 2 589 3 595 Mean 590 Orbscan 1st Visit 1 572 2 588 3 595 Mean 585 Orbscan 2nd Visit 1 602 2 587 3 588 Mean 592 Ultrasound 1st Visit 1 616 2 612 3 616 Mean 615 Ultrasound 2nd Visit 1 598 2 598 3 608 Mean 601

Table 46: Demographics and raw pachymetry in μm for subject 41

Age in years 36 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 22 yrs Rx Sphere -6.00 Cylinder -3.50 Axis 170 K's K 47.25 Axis 16 K 50.87 Axis 106 Visante 1st Visit 1 485 2 481 3 486 Mean 484 Visante 2nd Visit 1 488 2 494 3 489 Mean 490 Orbscan 1st Visit 1 445 2 424 3 407 Mean 425 Orbscan 2nd Visit 1 445 2 424 3 407 Mean 425 Ultrasound 1st Visit 1 523 2 522 3 537 Mean 527 Ultrasound 2nd Visit 1 515 2 501 3 508 Mean 508

Table 47: Demographics and raw pachymetry in μm for subject 42

Age in years 57 Gender Female Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 34 yrs Rx Sphere -1.62 Cylinder -1.62 Axis 59 K's K 46.5 Axis 32 K 53.87 Axis 122 Visante 1st Visit 1 481 2 487 3 477 Mean 482 Visante 2nd Visit 1 470 2 489 3 473 Mean 477 Orbscan 1st Visit 1 486 2 510 3 507 Mean 501 Orbscan 2nd Visit 1 504 2 492 3 495 Mean 497 Ultrasound 1st Visit 1 549 2 545 3 548 Mean 547 Ultrasound 2nd Visit 1 547 2 542 3 542 Mean 544

Table 48: Demographics and raw pachymetry in μm for subject 43

100

Age in years 42 Gender Female Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 19 yrs Rx Sphere -2.00 Cylinder -1.25 Axis 95 K's K 46.87 Axis 112 K 44.75 Axis 22 Visante 1st Visit 1 463 2 472 3 492 Mean 476 Visante 2nd Visit 1 487 2 476 3 469 Mean 477 Orbscan 1st Visit 1 425 2 441 3 431 Mean 432 Orbscan 2nd Visit 1 430 2 421 3 420 Mean 424 Ultrasound 1st Visit 1 479 2 505 3 472 Mean 485 Ultrasound 2nd Visit 1 499 2 510 3 506 Mean 505

Table 49: Demographics and raw pachymetry in μm for subject 44

101

Age in years 29 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 10 yrs Rx Sphere -1.75 Cylinder -3.62 Axis 1 K's K 43.62 Axis 5 K 52.37 Axis 95 Visante 1st Visit 1 515 2 510 3 512 Mean 512 Visante 2nd Visit 1 523 2 517 3 517 Mean 519 Orbscan 1st Visit 1 497 2 510 3 518 Mean 508 Orbscan 2nd Visit 1 507 2 511 3 525 Mean 514 Ultrasound 1st Visit 1 561 2 537 3 542 Mean 547 Ultrasound 2nd Visit 1 550 2 565 3 564 Mean 560

Table 50: Demographics and raw pachymetry in μm for subject 45

102

Age in years 27 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 15 yrs Rx Sphere -9.37 Cylinder -1.50 Axis 45 K's K 49.37 Axis 38 K 53.5 Axis 128 Visante 1st Visit 1 496 2 498 3 502 Mean 499 Visante 2nd Visit 1 508 2 512 3 500 Mean 507 Orbscan 1st Visit 1 517 2 525 3 511 Mean 518 Orbscan 2nd Visit 1 509 2 509 3 495 Mean 504 Ultrasound 1st Visit 1 527 2 506 3 510 Mean 514 Ultrasound 2nd Visit 1 473 2 486 3 484 Mean 481

Table 51: Demographics and raw pachymetry in μm for subject 46

103

Age in years 51 Gender Male Race Caucasian Cornea type Keratoconus Duration after Lasik or with Keratoconus 26 yrs Rx Sphere -1.50 Cylinder 0.00 Axis 0 K's K 42 Axis 12 K 42.75 Axis 102 Visante 1st Visit 1 560 2 554 3 560 Mean 558 Visante 2nd Visit 1 565 2 556 3 549 Mean 557 Orbscan 1st Visit 1 534 2 547 3 539 Mean 540 Orbscan 2nd Visit 1 544 2 543 3 523 Mean 537 Ultrasound 1st Visit 1 589 2 587 3 591 Mean 589 Ultrasound 2nd Visit 1 584 2 592 3 586 Mean 587

Table 52: Demographics and raw pachymetry in μm for subject 47

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