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CLINICAL SCIENCE

Central Corneal Thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachymetry Erica Brugin, MD,* Alessandra Ghirlando, MD,* Catia Gambato, MD,* and Edoardo Midena, MD*†

Purpose: To compare the repeatability and validity of corneal pachymetry by a corneal confocal microscope with a z-axis adapter (Confoscan 4.0 with z-ring adapter: z-CS4) versus ultrasound (US) pachymetry in the measurement of central corneal thickness (CCT).

Methods: CCT in 44 eyes of 44 subjects was determined with z-CS4. Z-CS4 exams were used to estimate the repeatability of thickness measurement by z-ring adapter for this confocal microscope. Intraclass Correlation Coefficient (ICC) between two different z-CS4 users was also determined. CCT in the same 44 eyes was determined with US pachymetry and measurements were compared with z-CS4 CCT.

Results: Z-CS4 CCT showed high intrainstrument reproducibility (ICC = 0.989; 95%CI 0.982-0.993; P , 0.0001). Mean difference among three CCT consecutive measures, in the same eye, was 0.8 6 11.1 mm. High correlation was found between two users (ICC = 0.896; 95%IC 0.830-0.937; P , 0.0001). Z-CS4 CCT showed high correlation with US pachymetry (ICC = 0.921; 95%CI 0.851-0.958; P , 0.0001). Mean corneal thickness determined was statistically different with the two methods (US: 512.6 6 65.8 mm; z-CS4: 487.8 6 60.1 mm; P , 0.0001). Conclusion: Z-CS4 seems an accurate, noninvasive and reproducible technique for CCT evaluation and confirms that central cornea is thinner when measured with confocal microscopy compared to ultrasounds. Key Words: corneal pachymetry, ultrasound pachymetry, corneal confocal microscopy, z-ring adapter (Cornea 2007;26:303–307)

C

orneal thickness is a major factor for planning and monitoring photorefractive procedures and monitoring several corneal diseases.1–10 Techniques for measuring central corneal thickness (CCT) include optical pachymetry, ultrasound pachymetry, confocal microscopy, ultrasound

Received for publication April 4, 2006; revision received September 11, 2006; accepted November 3, 2006. From the *Department of Ophthalmology, University of Padova, Padova, Italy; and the †Fondazione GB Bietti, IRCCS, Roma, Italy. The authors state that they have no proprietary interest in the products named in this article. Reprints: Edoardo Midena, Department of Ophthalmology, University of Padova, Via Giustiniani 2, 35128 Padova, Italy (e-mail: edoardo.midena@ unipd.it). Copyright Ó 2007 by Lippincott Williams & Wilkins

biomicroscopy, optical ray path analysis or scanning-slit corneal topography, and optical coherence tomography.11–20 Ultrasound pachymetry is the current standard for corneal thickness measurement.21–24 In vivo corneal confocal microscopy (CCM) is a diagnostic technique of the cornea that allows the visualization of a small area (;340 3 255 mm) of the cornea resolved in thin confocal slices.25,26 CCM allows one to analyze and record the different corneal layers with magnification up to 31000. Unfortunately, current corneal confocal microscope configuration is unable to provide completely accurate quantification along the eye z-axis because of eye movements in an antero-posterior direction. Recently, a corneal confocal microscope with a z-ring adapter (z-ring Confoscan 4.0; Nidek Technologies, Padova, Italy) claiming to accurately measure corneal layer distance along the z-axis has been introduced in clinical practice. The aim of this study was twofold: to test z-ring CCM for repeatability and accuracy and to compare corneal pachymetry performed by confocal microscope with z-ring adapter versus standard ultrasound pachymetry.

MATERIALS AND METHODS This cross-sectional study included 44 eyes of 44 consecutive subjects referred to the Laboratory of Advanced Ocular Diagnostic of the Department of Ophthalmology of the University of Padova. All subjects had a complete ophthalmologic examination including anterior segment biomicroscopy, funduscopy, corneal pachymetry, and corneal topography. The last procedure was performed primarily to exclude keratoconus. CCT was measured by standard ultrasound pachymetry and CCM. One eye of each subject was randomly selected for measurement, and the study eye was assigned to ultrasound and confocal microscopy pachymetry in randomized order, according to a 2 3 2 Latin Square to ensure complete balance between instruments’ evaluation. Informed consent was obtained from all subjects after the nature and purpose of the study were explained. All measurements were taken in late morning/early afternoon, when corneal thickness is considered stable.27 The interval between measurements was selected to maintain standard condition for both measurements (the 2 tests were performed within 6 hours).28 In vivo CCM was performed by using z-ring Confoscan 4.0, a scanning slit confocal microscope equipped with an Achroplan (Zeiss, Oberkochen, Germany) nonapplanating 340 immersion objective lens designed for full-thickness examination of the cornea, with a working distance of 1.92 mm and a motorized focusing device. Confoscan 4.0 is now

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equipped with a z-ring adapter system (z-CS4; Fig. 1). The z-ring adapter is an optomagnetic sensor that makes the examined eye integral with the tip of the confocal microscope during the examination. Therefore, with the z-ring sensor, the confocal microscope tip position turns out to be fixed in regard to the examined cornea, compensating for eye movements along the z-axis. The z-ring adapter is made of a part settled on the optical tip and of a removable part including a metallic stand and a contact ring in polymethylmethacrylate (PMMA; external diameter, 11 mm; internal diameter, 9 mm). The z-ring adapter is related to the cornea through a thin layer of polyacrylic gel, and during the scan, it is able to keep uniform pressure (maximum pressure of 30 mm Hg) on the cornea by proper sensor. Reliability of measures by z-CS4 was previously checked with a set of PMMA contact lenses with known thickness between 350 and 650 mm, and differences were within 65 mm (E. Midena, unpublished data). The center of the cornea was studied in all examinations. Before the examination, a drop of topical anesthetic 0.4% oxybuprocaine chlorohydrate (Novesina; Novartis Farma, Varese, Italy) was instilled in the lower conjunctival fornix of the eye to reduce blinking. The patient was seated in front of the microscope, with the aid of a chin rest and a forehead support, while fixating, with the examined eye, on a bright blue target inside the instrument to minimize eye movement during the examination. One drop of 0.2% polyacrylic gel (Viscotirs gel; Ciba Vision Ophthalmics, Venezia, Italy) was applied onto the objective tip to serve as an immersion fluid. Baseline macrometric alignment to the corneal apex was made manually by the operator, who moves the z-ring adapter to the apex of cornea. This procedure is automatically completed by a micrometric motor-driven system. The focal plane is automatically moved to reach the anterior chamber and begins scanning and recording of corneal images. The images are

FIGURE 1. Schematic drawing showing the z-ring adapter system: The constant pressure system continuously records the pressure of the objective lens onto the eye (which may change depending on the eye movement), and the software automatically moves the optical head forward or backward. This feedback allows to maintain a constant contact pressure. In the bottom right-hand, a real picture of the z-ring adapter. (M: motor)

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always taken from the endothelium to the corneal epithelium.23 Image intensity depth profiles are generated from confocal microscope video recordings by averaging the pixel intensity in the center of each consecutive video frame image and plotting data as a function of the z-depth in the z-scan curve. Data obtained from these scans are averaged and used for subsequent calculations. Every point in the z-scan curve is quantified in international units (IUs). Because of the z-ring adapter, all points on the z-scan curve are directly correlated to high-resolution images, and the exact z-axis position of specific tissue landmarks (ie, the surface epithelium, subepithelial nerve plexus, endothelium) are considered to calculate the distance between individual corneal layers. The confocal microscopy z-scan profile shows 2 peaks (identified in the z-scan curve) corresponding to major reflection originating from the endothelium (first peak) and superficial epithelial cells (second peak; Fig. 2). Whereas the first peak is sharp and well defined, the second one appears as a wide curve corresponding to several epithelial images. Corneal thickness was defined as the distance between the first peak (endothelium) and the last clear and centered frame of all epithelial images within the second peak. Each corneal confocal microscope examination was preplanned with the following parameters: a z-axis range of movement of 900 mm (3 passes for each complete corneal scan), thus giving a theoretical z-axis distance between images in the scans of 7 mm. Z-CS4 acquires 350 images per examination at a rate of 25 frames per second. Therefore, capture time is 14 seconds. The speed of the image plane at the cornea is approximately 72 mm/s. To define the accuracy of corneal confocal pachymetry by the z-ring adapter system, the 3 CCTs for each examination were

FIGURE 2. Corneal confocal microscopy frame with z-scan curve. Z-scan curve (bottom): the first peak corresponds to the endothelium (high reflectivity) peak; the second peak corresponds to the last high quality epithelium image. q 2007 Lippincott Williams & Wilkins

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analyzed. To determine interobserver repeatability of z-ring corneal confocal pachymetry, all subjects were tested by 2 independent and masked operators, both experienced in the use of the corneal confocal microscope. Each cornea was also measured using an ultrasonic pachymeter (UP1000; Nidek, Gamagori, Japan), calibrated by the manufacturer. This instrument uses an acoustic index of 1640 m/s to determine corneal thickness. One drop of 0.4% oxybuprocaine chlorohydrate (Novesina; Novartis Farma) was instilled in the conjunctival fornix. The probe was lightly touched to the central corneal 5 times, a thickness measurement was recorded for each touch, and these measurements were averaged. Z-CS4 uses an autoalignment system, and recording starts when the tip of the objective lens is aligned with the center of the cornea. The operator is able to document this phase by using the real-time monitor image that seems perfectly centered without lateral dark bands. The system is non–operator dependent. For ultrasound pachymetry examination, patients were asked to fixate on a small target with the fellow eye, and the tip of the ultrasound probe was manually centered on the pupil visually, as done in previous studies. The intraclass correlation coefficient (ICC) and its 95% confidence interval (95% CI) were used to determine the reliability of the confocal z-CS4 readings. For the interobserver reliability assessment and the comparison of z-CS4 with ultrasound pachymetry, a Bland–Altman graph was also evaluated. Paired student t test was used to evaluate the statistical difference between corneal thickness as measured by the 2 operators and by the 2 instruments. To determine the interobserver repeatability of z-CS4, the average of the 3 peak values obtained on each examination by each operator was used (44 by 2 operators). Intrainstrument repeatability of the CCT measurements performed by z-CS4 was tested comparing the 3 peak values obtained from the scans of each examined eye by both operators (3 values by 88 exams). To evaluate the agreement between z-CS4 and ultrasound pachymetry (‘‘gold standard’’) in measuring corneal thickness, the average of the 6 values obtained from the z-CS4 examinations of observer 1 and 2 and ultrasound values were used (44 values by 2 instruments). All statistical analyses were performed on a computer using a SPSS software package (SPSS for Windows, version 12.0; SPSS, Chicago, IL). Values of #0.05 (2-sided) were considered to indicate statistical significance.

CCT: Z-Ring CCM Versus Ultrasound Pachymetry

CI, 0.982–0.993; P , 0.0001). The mean difference among 3 CCT measurements in the same eye was 0.8 6 11.1 mm. The mean difference in CCT between scans taken by 2 different operators with the z-CS4 was not statistically significant (first operator: 500.1 6 72.0 mm; second operator: 496.2 6 67.3 mm; P = 0.356). Figure 3 shows interobserver repeatability data (ICC = 0.896; 95% CI, 0.830–0.937; P , 0.0001). The comparison of CCT between ultrasound pachymetry and z-CS4 in 44 eyes showed a significantly high ICC of 0.921 (95% CI, 0.851–0.958; P , 0.0001). The difference between the mean values of corneal thickness calculated using the CCM and ultrasonic pachymeter was 24.8 mm. In fact, mean corneal thickness determined with the ultrasound pachymeter was 512.6 6 65.8 versus 487.8 6 60.1 mm with the z-CS4 (paired t test; P , 0.0001; Fig. 4).

DISCUSSION The measurement of CCT has become increasingly important in ophthalmic practice. Refractive surgery is routinely planned according to preoperative measurement of CCT,8 and accurate determination of intraocular pressure may need to be modified according to CCT.5,7,9,10 Currently, the most used clinical method to measure CCT is ultrasound pachymetry. Ultrasound pachymeters are portable, objective, and simple-to-use instruments. Recent studies have documented that this method has a high degree of intraoperator, interoperator, and intrainstrument reproducibility.17–19,22,23 However, ultrasound pachymeter repeatability, even if good, varies from one instrument to another. Moreover, ultrasound pachymetry has the disadvantage that the placement of the probe exactly on the center of the cornea is crude and operator dependent. This method also requires direct cornea–probe contact.21 Mild patient discomfort and risk for infection are

RESULTS Forty-four eyes of 44 subjects (21 men and 23 women; mean age, 34 6 7 years; range, 22–49 years) were examined by ultrasound pachymeter and CCM with z-ring adapter (z-CS4). Mean corneal thickness quantified by these instruments was calculated for all subjects. Twenty of 44 eyes had previously undergone (.5 years) refractive surgery. CCM images, examined by an expert operator, never showed pathologic corneal reflectivity (normal corneal reflectivity: ,30 IU, as previously reported by Gambato et al29). The intrainstrument reproducibility of CCT measurements performed by the z-CS4 was ICC = 0.989 (95%

FIGURE 3. Central corneal thickness measured by confocal microscopy (Z-CS4) by two different operators. (Bland–Altman graph) Data are reported in microns (Intraclass correlation coefficient = 0.896; 95% CI 0.830-0.937; P , 0.0001).

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FIGURE 4. Central corneal thickness measured by confocal microscopy (Z-CS4) vs ultrasound pachymetry. (Bland–Altman graph) Data are reported in microns. (Intraclass correlation coefficient = 0.921; 95% CI 0.851-0.958; P , 0.0001. Mean central corneal thickness 6 SD: z-ring CS4 = 487.8 6 60.1 mm versus ultrasound = 512.6 6 65.8 mm; P , 0.0001).

additional concerns with a direct contact technique.21 A new instrument, the corneal confocal microscope, enables scanning of the different layers of the central cornea with magnification up to 31000 and analyzing individual corneal layers. This instrument allows central corneal analysis without direct cornea–instrument contact because of a thin layer of polyacrylic gel between the cornea and probe during the scan. Until now, the greatest limitation of CCM pachymetry compared with ultrasound pachymetry was the lack of precision because of the time required to scan through the cornea and the inevitable corneal movements during the scan (ie, the anterior–posterior motion caused by ocular pulse).1 Antero-posterior eye movements during the scan lead to an unpredictable error in the estimate of corneal thickness.1 The new z-ring adapter used with corneal confocal microscope Confoscan 4.0 has an eye–tip system that avoids the error produced by eye movements along the z-axis. Therefore, the corneal confocal microscope equipped with a z-ring allows calculation of the distance between corneal layer images and measurement of corneal thickness. The corneal thickness of a consecutive series of referred patients was measured with a z-CS4 and compared with a standard ultrasound pachymeter. Our data show the high intrainstrument repeatability and the high interobserver repeatability of the z-CS4. Our data also show high validity of the z-CS4 in reading pachymetric values compared with the ultrasound pachymeter. Therefore, the corneal confocal microscope with z-ring adapter seems to be a diagnostic technique that allows an accurate, reproducible measurement, without direct contact of the tip of the lens to the center of the cornea. McLaren et al1 compared CCT determined by ultrasonic and corneal confocal scanning slit (without z-ring adapter) pachymetry. Mean

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thickness measured by calibrated CCM was statistically lower than with ultrasonic pachymetry measurements.1 Javaloy et al3 found similar corneal thickness values with conventional ultrasound, scanning-slit corneal topography, and CCM. Different results may depend on the use of 2 different corneal confocal microscopes, the different ultrasound pachymeters, or the different method used to define corneal confocal pachymetry. Moreover, the confocal microscope used by Javaloy et al3 was not calibrated. McLaren at al1 concluded that an ultrasound pachymeter is suitable for measuring corneal thickness if relative changes in thickness are relevant and if the same instrument is used throughout the study. However, if true thickness is needed to make clinical decisions or to compare with other studies, the accuracy of a particular ultrasound pachymeter should be evaluated by an independent measurement. The difference between ultrasound and confocal microscope pachymetry suggests that verification of clinical ultrasonic pachymeters should be revised. McLaren et al1 also reported that their method of confocal microscopy pachymetry was not a valid alternative, because its reliability was limited by corneal motion in the antero-posterior direction. Our method, which uses a z-ring adapter, avoids this error. Our data confirm the difference between ultrasound and confocal microscopy pachymetry, but this difference is lower in our study than that in the report by McLaren et al1 (24.8 vs. ;38 mm). Lower CCT differences may depend on the use of the z-ring in this study, because other variables, such as corneal refractive index and corneal radius of curvature, have already been shown to be irrelevant. Many studies have reported consistent differences when the same cornea is measured with different approaches.11–15,18–20 We believe that z-CS4 measurement approaches real corneal thickness, because this technique overwhelms the major limitation of CCM. When it is necessary to accurately determine CCT for clinical intervention, it seems relevant to improve current techniques. CCM with the z-ring adapter may represent a new and valid approach. REFERENCES 1. McLaren JW, Nau CB, Erie JC, et al. Corneal thickness measurement by confocal microscopy, ultrasound, and scanning slit methods. Am J Ophthalmol. 2004;137:1011–1020. 2. Holden BA, Mertz GW, McNally JJ. Corneal swelling response to contact lenses worn under extended wear conditions. Invest Ophthalmol Vis Sci. 1983;24:218–226. 3. Javaloy J, Vidal MT, Villada JR, et al. Comparison of four corneal pachymetry techniques in corneal refractive surgery. J Refract Surg. 2004;20:29–34. 4. Pflugfelder SC, Liu Z, Feuer W, et al. Corneal thickness indices discriminate between keratoconus and contact-lens induced corneal thinning. Ophthalmology. 2002;109:2336–2341. 5. Herndon LW, Weizer JS, Stinnett SS. Central corneal thickness as a risk factor for advanced glaucoma damage. Arch Ophthalmol. 2004;122: 17–21. 6. Medeiros FA, Sample PA, Weinreb RN. Corneal thickness measurements and frequency doubling technology perimetry abnormalities in ocular hypertensive eyes. Ophthalmology. 2003;110:1903–1908. 7. Medeiros FA, Sample PA, Weinreb RN. Corneal thickness measurements and visual function abnormalities in ocular hypertensive patients. Am J Ophthalmol. 2003;135:131–137. 8. Price FW Jr, Koller DL, Price MO. Central corneal pachymetry in patients undergoing laser in situ keratomileusis. Ophthalmology. 1999;106:2216– 2220.

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