September 2011
Volume 52, Issue 10
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Cornea  |   September 2011
Normative Database for Corneal Backscatter Analysis by In Vivo Confocal Microscopy
Author Affiliations & Notes
  • Toine Hillenaar
    From the Rotterdam Ophthalmic Institute (ROI), Rotterdam, The Netherlands;
    the Cornea and External Disease Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands; and the
  • Roger H. H. Cals
    the Cornea and External Disease Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands; and the
  • Paul H. C. Eilers
    Department of Biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands.
  • René J. Wubbels
    From the Rotterdam Ophthalmic Institute (ROI), Rotterdam, The Netherlands;
  • Hugo van Cleynenbreugel
    the Cornea and External Disease Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands; and the
  • Lies Remeijer
    the Cornea and External Disease Service, The Rotterdam Eye Hospital, Rotterdam, The Netherlands; and the
  • Corresponding author: Toine Hillenaar, The Rotterdam Eye Hospital, PO Box 70030, 3000 LM Rotterdam, The Netherlands; t.hillenaar@oogziekenhuis.nl
Investigative Ophthalmology & Visual Science September 2011, Vol.52, 7274-7281. doi:10.1167/iovs.11-7747
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      Toine Hillenaar, Roger H. H. Cals, Paul H. C. Eilers, René J. Wubbels, Hugo van Cleynenbreugel, Lies Remeijer; Normative Database for Corneal Backscatter Analysis by In Vivo Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2011;52(10):7274-7281. doi: 10.1167/iovs.11-7747.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To ascertain the sex and age relatedness, diurnal variation, and repeatability of backscatter measurement in the normal human cornea.

Methods.: Seven corneal backscatter variants were measured by in vivo confocal microscopy (IVCM) in both normal eyes (n = 314) of 157 healthy subjects. These subjects were assigned to one or more of three groups. The sex and age relatedness of corneal backscatter were assessed in group 1 (n = 300), which comprised 75 men and 75 women evenly distributed over five age categories. To assess diurnal variation, eyes in group 2 (n = 40) were measured four times a day, at 3-hour intervals. The eyes in group 3 (n = 50) were examined four times a year to determine intersession repeatability. Intrasession repeatability was determined by performing all IVCM examinations in duplicate. Linear mixed models were used to assess the effects of sex, age, and time of measurement on corneal backscatter.

Results.: Mean corneal backscatter was 3.5% higher in men (P = 0.003). From the age of 50 years, backscatter increased significantly in the anterior stroma (P = 0.0003). A small but statistically significant diurnal variation was found in all seven backscatter variants (P < 0.01). The test–retest coefficient of variation of mean corneal backscatter was 5.3%, comprising intra- and intersession repeatability.

Conclusions.: Sex and time of measurement significantly affect corneal backscatter measured by IVCM, whereas age affects only backscatter in the anterior stroma. All three factors should be taken into account when conducting scientific research. For ophthalmic practice, the authors suggest ignoring these factors and propose a generalized normal range and minimum detectable change for each backscatter variant.

In the past decade, the number of imaging techniques for in vivo assessment of the human cornea has burgeoned. Whereas imaging techniques such as Scheimpflug photography and anterior segment optical coherence tomography have been implemented in the ophthalmic clinic, in vivo confocal microscopy (IVCM) has never become common practice. Despite its great potential, the clinical use of IVCM remains confined to the diagnosis of rare corneal degenerations and dystrophies and the differentiation of uncommon pathogens in infectious keratitis. 1 5 Only recently has a wider clinical application been introduced that exploits the ability of IVCM to detect endothelial involvement in order to monitor disease activity in inflammatory corneal processes. 6 In addition to evaluating the corneal endothelium, disease status may also be assessed by another feature of IVCM, corneal backscatter. 
Although corneal backscatter has been studied in normal eyes using IVCM, 7,8 fundamental data about the effects of sex, age, and diurnal variation are still lacking. Knowledge of these effects and information on the repeatability of backscatter measurements are essential when studying disease progression in pathologic processes of the cornea or the outcome of corneal surgery. The purpose of this study was to ascertain the effects of sex, age, and time of measurement on corneal backscatter and to assess its repeatability as measured by IVCM. 
Methods
Subjects
For the present study, the eyes of 157 subjects (n = 314) were enrolled and categorized into three groups (Table 1). Several subjects were assigned to more than one group. Group 1 (n = 300) was designed to assess the sex- and age-related variation of corneal backscatter. Subjects in this group were evenly distributed over five age categories; 20 to 29, 30 to 39, 40 to 49, 50 to 59, and 60 to 79 years. Each category consisted of 15 men and 15 women. We also used group 1 to ascertain the morphologic aspects of aging in the normal cornea. Since the results on this topic are beyond the scope of the current paper, they are described in a separate paper. Diurnal variation in corneal backscatter was assessed in group 2 (n = 40). Subjects in group 2 were examined four times a day at 3-hour intervals. Group 3 comprised 25 subjects (n = 50) who were examined four times a year at 3-month intervals, to determine intersession repeatability. To assess intersession repeatability, we assumed that backscatter in a normal cornea remained stable over this 1-year period. All IVCM examinations were performed in duplicate to determine intrasession repeatability. 
Table 1.
 
Group Characteristics
Table 1.
 
Group Characteristics
Group 1 Sex, Age Relatedness Group 2 Diurnal Variation Group 3 Intersession Repeatability
Subjects, n 150 20 25
Mean age, y 45 (20–79) 38 (20–59) 44 (27–67)
Men, n 75 7 8
Eyes, n 300 40 50
Sessions, n 1 4 4
Time 9:00–12:00 9:00, 12:00, 15:00, 18:00 9:00–12:00
Month 0 0 0, 3, 6, 9
IVCM + + +
US pachymetry + +
GAT IOP + +
Subjects neither had a history of systemic disease, nor used any medication known to affect corneal transparency. Other exclusion criteria were previous inflammation, infection, or allergic reaction of the eye; ocular surgery or trauma; or a history of contact lens wear. Before enrollment subjects were examined by slit lamp biomicroscopy, to confirm an intact corneal epithelium, a clear corneal stroma, and absence of ocular inflammatory processes. 
The protocol of this cross-sectional study was approved by our institutional review board and the local medical ethics committee and was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects. 
Corneal Backscatter Measurement
Corneal backscatter was measured by IVCM (Confoscan 4; Nidek Technologies, Padova, Italy), according to a previously described method. 6 Briefly, both subjects' eyes were given 1 drop of 0.4% oxybuprocaine (Ceban BV, Breda, The Netherlands) to ensure corneal anesthesia. An immersion gel (Vidisic; Dr. Mann Pharma, Berlin, Germany) was applied to the 40× objective lens. We used a z-ring adapter to enable backscatter measurement at different corneal depths and to reduce motion artifacts in the z-axis. After autoalignment, the central cornea was scanned in full-thickness mode with fixed device settings: 72% light intensity and a 6-μm scan step. With these settings, a scan contained three complete passes through the cornea. 
Because corneal backscatter analysis by IVCM is subject to large interinstrument differences, 9 we standardized our measurements with a standard turbidity suspension (AMCO Clear; GFS Chemicals, Inc., Powell, OH), after which backscatter was expressed in scatter units (SU). 8,9 To test for light intensity variations of the confocal microscope over the day, we examined a solid piece of polymethylmethacrylate (PMMA; Opal 040 Perspex GS, Lucite International Ltd., Southampton, UK) on four occasions during a day. The same PMMA slab was used for a weekly reference scan to obtain long-term standardization of backscatter measurement. Once a year, opacity changes in the PMMA slab were verified (AMCO Clear; GFS Chemicals, Inc.). During the study, the ambient light in the room was kept constant. 
For each measurement, seven variants of corneal backscatter were calculated (Fig. 1). Mean backscatter of the selected images of three passes through the cornea was used for backscatter values of the cornea, stroma, and the anterior, middle, and posterior thirds of the stroma. After determining the stromal boundaries (see the Appendix, Supplementary Material S1), these values were computed semiautomatically with a purpose-made algorithm (see the algorithm, Supplementary Material S2, and the algorithm manual, Supplementary Material S3). Manually determined mean peak values were used to express the epithelial valley (EV) and subepithelial peak (SP). Intra- and intersession repeatability were assessed for all seven variants of corneal backscatter. 
Figure 1.
 
Backscatter measurements by IVCM. The IVCM images are 425 × 320 μm, with the bar representing 50 μm. Six characteristic layers of a normal human cornea and their position on the z-scan curve: (A) corneal endothelium; (B) corneal stroma showing a large straight nerve fiber; (C) the anterior stroma, characterized by a higher keratocyte density compared to middle and posterior thirds of the stroma. 10 ; (D) subbasal nerve plexus; (E) basal epithelial cell layer; and (F) superficial epithelial cells. (a–g) Seven variants of corneal backscatter. For variants a to e, mean backscatter per image was calculated in three subsequent passes through the cornea. 9 Variants f and g represent mean peak values of the three subsequent passes in one scan. Backscatter of the (a) cornea, (b) stroma, (c) posterior third of the stroma, (d) middle third of the stroma, (e) anterior third of the stroma and of the (f) SP and (g) EV.
Figure 1.
 
Backscatter measurements by IVCM. The IVCM images are 425 × 320 μm, with the bar representing 50 μm. Six characteristic layers of a normal human cornea and their position on the z-scan curve: (A) corneal endothelium; (B) corneal stroma showing a large straight nerve fiber; (C) the anterior stroma, characterized by a higher keratocyte density compared to middle and posterior thirds of the stroma. 10 ; (D) subbasal nerve plexus; (E) basal epithelial cell layer; and (F) superficial epithelial cells. (a–g) Seven variants of corneal backscatter. For variants a to e, mean backscatter per image was calculated in three subsequent passes through the cornea. 9 Variants f and g represent mean peak values of the three subsequent passes in one scan. Backscatter of the (a) cornea, (b) stroma, (c) posterior third of the stroma, (d) middle third of the stroma, (e) anterior third of the stroma and of the (f) SP and (g) EV.
Covariates
Because diurnal variation in central corneal thickness (CCT) and intraocular pressure (IOP) may correlate with corneal backscatter, these possible covariates were measured simultaneously with the IVCM examinations in groups 1 and 2. We recorded CCT as the mean of 10 readings with ultrasound (US) pachymetry (model SP-3000; Tomey Ltd., Tokyo, Japan). Intraocular pressure, measured with Goldmann applanation tonometry, was always performed after IVCM, to avoid interference of fluorescent staining of the tear film with the backscatter measurements. Both CCT and IOP were performed under topical anesthesia with oxybuprocaine 0.4%. 
Statistical Analyses
P values were considered statistically significant when < 0.05 (SPSS for Windows, ver. 17.0; SPSS, Inc., Chicago, IL). To account for clustering of eyes within subjects, we used linear mixed models to assess sex and age relatedness (group 1) and diurnal variation of corneal backscatter (group 2). In the model for group 1, sex and eye were considered fixed factors, whereas age, CCT, and IOP were entered as covariates. The model for group 2 resembled the model for group 1 with the distinction that the covariate age was replaced with the fixed factor time. IOP was not included in the model for group 2, because it strongly correlated with time and caused collinearity of the statistical model. 
Intra- and intersession repeatability were expressed in relative terms as coefficient of variation (COV) and in absolute terms as coefficient of repeatability (COR). The COV was defined as the within-subject SD (SDW) divided by the mean backscatter and was expressed as a percentage, whereas the COR was defined as 1.96(√2 × SDW). 11 Intrasession repeatability was calculated on the basis of 584 duplicate backscatter measurements. These duplicate measurements were also used to test whether subjects got accustomed to the IVCM examinations, which in turn may have resulted in better quality backscatter measurements. We analyzed this so-called “learning effect” by comparing all first measurements of each eye to the second measurements of that eye, using a paired Student's t-test. Furthermore, we used equation 1 to calculate the test–retest coefficient of variation (COVT) for corneal backscatter on the basis of intrasession (COVA) and intersession (COVB) components:    
After considering the effects of sex, age, and time of measurement on corneal backscatter and assessing its repeatability, we constructed a generalized normal range for corneal backscatter. For this purpose, we applied a logarithmic transformation to acquire a between-subject SD accounting for skewness of the data. 12 As normal range, we chose a 99% rather than a 95% reference interval because we expected our study population to comprise only supernormal corneas, as contact lens wearers and subjects who had undergone cataract surgery were not enrolled in the study. By taking the antilog, the normal range was transformed back to the original scale. 
To identify clinically relevant change, the difference between two measurements within one subject should be larger than the difference explained by measurement error. 13 This minimum detectable change at the 95% confidence level (MDC95%) is defined by the COR. Because the COR varies with the amount of backscatter, 9 we expressed the MDC95% as a percentage. By using equation 2 to compute the MDC95%, we also accounted for diurnal variation of corneal backscatter:    
Results
Mean corneal backscatter was 3.5% higher in the men than in the women (regression coefficient = 54.4 ± 36.3, P = 0.003; Table 2). Two outliers were seen at the higher end of the age spectrum (Fig. 2A, arrows). On closer inspection of these two outliers, the IVCM images showed abnormal depositions in the stroma of both corneas and epithelial deviations in one cornea. After exclusion of the outliers from statistical analysis, the influence of age on mean stromal backscatter was borderline significant (P = 0.05), because of strong age relatedness of backscatter in the anterior stroma (P = 0.0003; Table 3). Backscatter in the anterior stroma decreased slightly until 50 years of age, but increased significantly thereafter (Fig. 2B). The measured corneal backscatter correlated negatively with CCT, but was not correlated with IOP (Figs. 2C, 2D). We found no differences between right and left eyes. 
Table 2.
 
Sex Relatedness of Corneal Backscatter Measured by IVCM
Table 2.
 
Sex Relatedness of Corneal Backscatter Measured by IVCM
Men Women ΔSEX (%) P
Cornea 1248 ± 149 1206 ± 96 3.5 0.003
Stroma 1106 ± 181 1057 ± 108 4.6 0.005
Anterior stroma 1145 ± 177 1117 ± 125 2.5 NS
Mid stroma 962 ± 171 925 ± 106 4.1 0.02
Posterior stroma 1211 ± 232 1130 ± 135 7.2 0.001
Epithelial valley 1044 ± 103 1024 ± 99 1.9 NS
Subepithelial peak 1579 ± 252 1446 ± 177 9.2 0.0002
Figure 2.
 
Age relatedness of corneal backscatter. (A) Mean corneal backscatter remained relatively stable with increasing age. Two outliers were seen at the higher end of the age spectrum (arrows). (B) Backscatter in the anterior third of the stroma per age category. After an initial decrease up to 50 years of age, backscatter in the anterior stroma showed an increase in the last two age categories. The last age category spanned 20 years instead of 10 years and was, for the purpose of this figure, divided into two decades, which comprised n = 42* and n = 18** eyes. Error bars represent 95% confidence intervals. (C) CCT correlated negatively with mean corneal backscatter (regression coefficient = −1.21 ± 0.25, P < 0.0001). (D) IOP did not correlate with mean corneal backscatter.
Figure 2.
 
Age relatedness of corneal backscatter. (A) Mean corneal backscatter remained relatively stable with increasing age. Two outliers were seen at the higher end of the age spectrum (arrows). (B) Backscatter in the anterior third of the stroma per age category. After an initial decrease up to 50 years of age, backscatter in the anterior stroma showed an increase in the last two age categories. The last age category spanned 20 years instead of 10 years and was, for the purpose of this figure, divided into two decades, which comprised n = 42* and n = 18** eyes. Error bars represent 95% confidence intervals. (C) CCT correlated negatively with mean corneal backscatter (regression coefficient = −1.21 ± 0.25, P < 0.0001). (D) IOP did not correlate with mean corneal backscatter.
Table 3.
 
Age-Related Changes of Corneal Backscatter Measured by IVCM
Table 3.
 
Age-Related Changes of Corneal Backscatter Measured by IVCM
20–29 y 30–39 y 40–49 y 50–59 y 60–79 y Max ΔAGE (%) P
Cornea 1245 ± 100 1217 ± 97 1187 ± 106 1204 ± 113 1257 ± 123 5.9 NS
Stroma 1091 ± 114 1068 ± 118 1039 ± 119 1066 ± 125 1114 ± 144 7.2 0.05
Anterior stroma 1103 ± 110 1101 ± 131 1097 ± 146 1143 ± 135 1190 ± 148 8.5 0.0003
Mid stroma 958 ± 115 939 ± 118 903 ± 110 928 ± 115 960 ± 139 6.3 NS
Posterior stroma 1212 ± 162 1165 ± 143 1117 ± 143 1126 ± 172 1193 ± 180 8.5 NS
Epithelial valley 1085 ± 119 1017 ± 97 1008 ± 91 1014 ± 77 1039 ± 85 7.6 NS
Subepithelial peak 1520 ± 221 1459 ± 166 1457 ± 227 1545 ± 191 1560 ± 254 7.1 NS
All seven variants of corneal backscatter showed a small (≤4%), but statistically significant diurnal variation (Table 4). CCT measured with ultrasound remained stable over the day, whereas IOP changed significantly (by up to 14%; Fig. 3). The diurnal variation in IOP was negatively correlated with the change in corneal backscatter (regression coefficient = −7.0 ± 5.0, P = 0.006). 
Table 4.
 
Diurnal Variation of Corneal Backscatter Measured by IVCM
Table 4.
 
Diurnal Variation of Corneal Backscatter Measured by IVCM
9 h 12 h 15 h 18 h Max ΔDAY (%) P
Cornea 1220 ± 106 1238 ± 108 1253 ± 111 1250 ± 102 2.7 0.002
Stroma 1058 ± 119 1073 ± 119 1088 ± 120 1088 ± 115 2.8 0.0001
Anterior stroma 1085 ± 146 1108 ± 152 1122 ± 149 1114 ± 143 3.4 0.001
Mid stroma 924 ± 106 933 ± 101 948 ± 107 949 ± 103 2.7 0.0004
Posterior stroma 1165 ± 141 1179 ± 148 1194 ± 145 1200 ± 139 3.0 0.001
Epithelial valley 1053 ± 103 1081 ± 124 1087 ± 112 1092 ± 105 3.7 0.003
Subepithelial peak 1480 ± 211 1508 ± 218 1540 ± 231 1525 ± 210 4.1 0.01
Figure 3.
 
Diurnal variation of corneal backscatter. (A) Mean corneal backscatter varied significantly during the day: maximum variation 2.7%, P = 0.002. (B) CCT remained stable during the day. (C) Diurnal variation in IOP was exactly opposite to the change in corneal backscatter; maximum variation of IOP was 14.1%, P < 0.0001. Error bars represent 95% confidence intervals of the mean.
Figure 3.
 
Diurnal variation of corneal backscatter. (A) Mean corneal backscatter varied significantly during the day: maximum variation 2.7%, P = 0.002. (B) CCT remained stable during the day. (C) Diurnal variation in IOP was exactly opposite to the change in corneal backscatter; maximum variation of IOP was 14.1%, P < 0.0001. Error bars represent 95% confidence intervals of the mean.
With test–retest COVs around 5%, all seven backscatter variants had a very good repeatability (Table 5). Only the backscatter measurement of the posterior third of the stroma had a slightly higher test–retest COV: 7%. We found no difference between intra- and intersession repeatability. 
Table 5.
 
Repeatability of Backscatter Measured by IVCM
Table 5.
 
Repeatability of Backscatter Measured by IVCM
Intrasession Intersession COVT (%)
COV (%) COR COV (%) COR
Cornea 4.2 143 3.2 111 5.3
Stroma 3.8 114 3.2 98 5.0
Anterior stroma 4.0 127 3.5 111 5.3
Mid stroma 3.7 98 3.4 88 5.0
Posterior stroma 5.2 170 4.3 140 6.8
Epithelial valley 3.8 111 3.0 84 4.8
Subepithelial peak 4.0 169 3.6 155 5.4
When corneal backscatter is applied in ophthalmic practice, it is much more feasible to ignore the effects of sex, age, and time of measurement. Since these effects were relatively small, we constructed a generalized normal range and MDC95% for all seven backscatter variants (Table 6). 
Table 6.
 
Generalized Normal Range and MDC95% of Corneal Backscatter
Table 6.
 
Generalized Normal Range and MDC95% of Corneal Backscatter
Mean 99% Reference Interval MDC95% (%)
Cornea 1222 966–1534 16
Stroma 1075 796–1435 15
Anterior stroma 1126 816–1531 16
Mid stroma 937 673–1285 15
Posterior stroma 1162 811–1635 20
Epithelial valley 1032 802–1316 15
Subepithelial peak 1508 1033–2156 16
Our results were unaffected by change in light intensity of the confocal microscope over the day or during the study period. Also, we could not establish a “learning effect”, as the first measurements were no different from the second measurements of the same eye. 
Discussion
The present study reports on corneal backscatter measurement in the largest normative IVCM database to date. Corneal backscatter in a normal population depended on the sex of the subject and time of measurement, whereas the influence of age was confined to backscatter in the anterior stroma. All three factors should be taken into account in the design of a study that compares corneal backscatter measured by IVCM between patient groups. For detection and follow-up of corneal haze in individual patients, as is the case in ophthalmic practice, these factors have to be considered in another perspective. We suggest ignoring sex, age, and time of measurement when using corneal backscatter in ophthalmic practice, because the influence of these factors is much smaller than the variability of corneal backscatter between and within subjects. 
Corneal backscatter has been reported to be sex independent. 14 In contrast with our study, these measurements were performed under nonspecular conditions. Under such conditions, scattering is elicited by the collagen fibrils in the stroma. 15 Because corneal backscatter is dominated by reflections from the cell nuclei when measured under specular conditions by IVCM, our results cannot be compared with Olsen's findings. 14 The reason for the small sex difference in corneal backscatter measured by IVCM, remains unclear. Sex difference in CCT, which might be influenced by hormones, 16 could not have interfered with our results, because CCT was a covariate in our linear mixed model. The keratocyte nuclei, a major source of corneal backscatter measured by IVCM, are also unlikely to have caused this difference between the sexes, as keratocyte density has been reported to be sex-independent. 10,17 Another explanation is a potential difference in the stromal extracellular matrix between men and women. This theory is supported by the higher incidence of corneal arcus in men. 18  
In the literature, there is no consensus on the age relatedness of corneal backscatter. In a study using blue light on a modified slit lamp, Olsen 14 found corneal backscatter to increase with age. This finding was disputed by other groups, who used Scheimpflug imaging 19 or white light on a modified slit lamp 20 and found corneal backscatter to be independent of age. Since different wavelengths and scattering angles were used in these studies and because corneal backscatter depends on these factors, 21,22 neither direct comparison of these studies nor comparison with our results is appropriate. The age-related backscatter pattern we found in the corneal stroma (Fig. 2B), can be explained by looking at the age-related morphologic changes. In young adults, backscatter in the stroma is largely composed of backscatter from the keratocyte nuclei. Because the number of keratocyte nuclei decreases with age, 10,17,23 stromal backscatter also decreases in the first five decades of life. In subjects aged 50 years and above, we found an increase in microdots, especially in the anterior third of the stroma (data not shown). This increase of small particles, which may represent dysgenic or apoptotic cellular remnants such as lipofuscin granules, 24,25 probably caused the increase in stromal backscatter. 
In contrast with other studies, 14,20 we found corneal backscatter to correlate inversely with CCT. By using a confocal microscope instead of a slit lamp, we incorporated the specular reflection of the corneal endothelium into our backscatter measurements. The specular reflection induced a backscatter peak that slightly increased mean corneal backscatter. This effect of the specular reflection on mean corneal backscatter becomes larger in a thinner cornea, because in a thinner cornea, backscatter is averaged over fewer images. 
To our knowledge, diurnal variation of corneal backscatter has never been studied before. Other well-established cyclic factors such as CCT 26,27 and IOP 28 may have influenced our results. To minimize the effect of overnight swelling of the cornea, we started our measurements at least 2 hours after awakening. Having taken this precaution, in common with many other reports, 16 we found CCT to be stable during working hours. The characteristic diurnal variation of intraocular pressure we found is similar to that reported by others. 28,29 Because the diurnal variation of IOP was exactly opposite to the diurnal variation of corneal backscatter and because these two parameters were highly inversely correlated, it is possible that collinearity occurred. Even though collinearity suggests that the diurnal variation of IOP affected corneal backscatter, the two parameters may still be independent. After excluding light intensity variation and the so-called “learning effect” as confounding factors, a third potentially confounding factor remains. Repeated exposure of the cornea to preservatives in the anesthetic eye drop and the coupling gel may accelerate desquamation of the superficial epithelial cells, subsequently increasing epithelial backscatter. 9 Although we did not observe any such increase of bright superficial epithelial cells during the four measurement sessions, subtle effects of preservatives on corneal backscatter cannot be ruled out. 
The repeatability of IVCM backscatter measurement depends on the homogeneity and degree of transparency of the specimen that is examined. 9 When examining a patient, however, the major limiting factor is the motion artifacts in the z-axis that occur during the 12 seconds of image acquisition. Consequently, low repeatability was reported for IVCM backscatter measurement in normal corneas: intrasession COR = 8.2 gray levels, intersession COR = 15.5 gray levels (Jalbert I, et al. IOVS 2002;43:ARVO E-Abstract 1713). Intersession repeatability was even worse for patients after excimer laser photorefractive keratectomy: COV = 35%. 30 The much higher intra- and intersession repeatability we found (Table 5) by using a z-ring adapter for stabilization of the image acquisition cannot be directly compared with the previous reports, as these reports did not calibrate their data sufficiently or examined corneas of different transparency. Our results can be compared with and are similar to calibrated slit lamp based backscatter measurement in normal corneas: COV = 3% to 7%. 14,20 Yet, these modified slit lamps are not commercially available and have a much lower axial and lateral resolution than IVCM. On the other hand, IVCM requires a specially trained optometrist or ophthalmologist to operate the confocal microscope and to assess the morphologic aspects of the corneal layers. Also, the quality of backscatter measurement by IVCM depends largely on the experience of the operator. To reduce interexaminer differences due to the subjective demarcation of the stromal boundaries, we protocolized the backscatter measurements (Appendix, Supplementary S1). Using this protocol, we found that the posterior stroma was harder to demarcate than the anterior stroma. This resulted in a slightly higher COV for the posterior stroma than for the other backscatter variants. 
Our findings imply that IVCM studies on corneal backscatter should account for the effects of sex, age, and time of measurement. For corneal backscatter to be used in ophthalmic practice, however, these effects on corneal backscatter should be contextualized. We suggest that the patient's sex and age be ignored, because their effects on the backscatter variants were smaller than the relative between-subject SDs. When the patient's sex and age are ignored, a generalized normal range for corneal backscatter may be used to detect corneal haze. The mean backscatter values of this normal range (Table 6) are somewhat higher than those reported by McLaren et al. 8 The origin of this disparity remains unclear and exemplifies the importance of a uniform calibration method as well as the need for a universal reference standard that is stable over time. 9  
Diurnal variation affects backscatter analysis only when corneal backscatter is monitored within a patient. Our finding that maximum diurnal variation for corneal backscatter was smaller than the test–retest COV (2.7% versus 5.3%) means that diurnal variation cannot reliably be observed in an individual patient. Nevertheless, diurnal variation should be taken into account when considering improvement or progression of corneal haze. By slightly overestimating the MDC95%, a clinically relevant change in corneal backscatter can still be detected when a follow-up visit is scheduled at a different time as the first visit. 
Some issues should be considered before corneal backscatter is applied in ophthalmic practice. Because IVCM uses a magnification up to 500 times, only 0.14% of the corneal surface is imaged. 6 As a result, positional repeatability is low. In our opinion, corneal haze can be monitored with sufficient repeatability only if the central cornea is imaged and if the corneal haze is more or less homogeneous. When these conditions are met, corneal disorders may be monitored by mean corneal backscatter, whereas the other six backscatter variants may be used for specific purposes. For example, backscatter of the basal epithelial cell layer (EV) can be used to assess corneal hydration (Figs. 4A, 4B). 31 The EV may prove more sensitive than CCT in the detection of corneal edema, and therefore may be an important parameter in the evaluation of the cornea in Fuchs endothelial dystrophy, before cataract surgery. Likewise, subepithelial fibrosis in Fuchs endothelial dystrophy may be staged with the SP, to estimate the effect of Descemet stripping automated endothelial keratoplasty (DSAEK) on the postoperative best corrected visual acuity (Figs. 4C, 4D). Mean stromal backscatter and subdivision into anterior, middle, and posterior thirds may be used to monitor the inflammatory process in herpetic stromal keratitis (Figs. 4E, 4F). The powerful combination of corneal backscatter measurement with morphologic assessment of the corneal layers may improve treatment strategies in this complex chronic disease. 
Figure 4.
 
Future applications of corneal backscatter measurement. Images are 425 × 320 μm, with the bar representing 50 μm. (A, B) The EV can be used to objectively assess corneal hydration. 31 (A) The EV was 3792 SU in a patient with Fuchs endothelial dystrophy before DSAEK. (B) One month after DSAEK, corneal edema had diminished in the same patient: 1170 SU. (C, D) Subepithelial fibrosis may be assessed with the SP. (C) Characteristic reticular meshwork of subepithelial fibrosis in Fuchs endothelial dystrophy. (D) Exactly the same location was imaged in the same patient 6 months after DSAEK. The shape of the reticular meshwork had remained completely stable. Nevertheless, the SP declined from 5268 to 1984 SU. (E, F) Backscatter of the anterior, middle, and posterior parts of the stroma and their overall mean, may be used to monitor the inflammatory process in herpetic stromal keratitis. (E) During a recurrence, the stroma of this patient with herpes simplex keratitis showed an increase in backscatter: 3189 SU. (F) Mean stromal backscatter had returned to normal in the same patient 1 year after recurrence of the immune stromal keratitis: 1226 SU. All images (AF) were acquired with fixed light intensity of 72%, after calibration of backscatter analysis.
Figure 4.
 
Future applications of corneal backscatter measurement. Images are 425 × 320 μm, with the bar representing 50 μm. (A, B) The EV can be used to objectively assess corneal hydration. 31 (A) The EV was 3792 SU in a patient with Fuchs endothelial dystrophy before DSAEK. (B) One month after DSAEK, corneal edema had diminished in the same patient: 1170 SU. (C, D) Subepithelial fibrosis may be assessed with the SP. (C) Characteristic reticular meshwork of subepithelial fibrosis in Fuchs endothelial dystrophy. (D) Exactly the same location was imaged in the same patient 6 months after DSAEK. The shape of the reticular meshwork had remained completely stable. Nevertheless, the SP declined from 5268 to 1984 SU. (E, F) Backscatter of the anterior, middle, and posterior parts of the stroma and their overall mean, may be used to monitor the inflammatory process in herpetic stromal keratitis. (E) During a recurrence, the stroma of this patient with herpes simplex keratitis showed an increase in backscatter: 3189 SU. (F) Mean stromal backscatter had returned to normal in the same patient 1 year after recurrence of the immune stromal keratitis: 1226 SU. All images (AF) were acquired with fixed light intensity of 72%, after calibration of backscatter analysis.
In conclusion, this large, normative IVCM database enabled us to identify sex and time of measurement as significant factors in corneal backscatter measurement. Age is a less significant factor, as its influence is confined to backscatter in the anterior stroma. For research purposes, all three factors should be taken into account, whereas for use in ophthalmic practice, we suggest incorporating the effects of sex and age into the normal range for corneal backscatter and accounting for diurnal variation in the definition for improvement or progression of corneal haze. Such a generalized normal range and minimum detectable change for each backscatter variant is easily accessible in a clinical setting. However, before backscatter measurement can be used to detect and monitor pathologic processes in the cornea, further research is needed. Our normative database may serve as a reference for future studies on the clinical value of corneal backscatter measurement by IVCM. 
Supplementary Materials
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The authors thank Sietske Huiskens and Elma Bras (Rotterdam Ophthalmic Institute) for excellent technical assistance, Netty Dorrestijn (Rotterdam Ophthalmic Institute) for organizing the referral of study subjects, and Tom van den Berg (Netherlands Institute for Neuroscience) for his helpful advice and suggestions in the preparation of this manuscript. 
Footnotes
 Supported by the Research Foundation SWOO Flieringa, Rotterdam; The Dutch Cornea Foundation, Rotterdam; and the OOG Foundation, 's Gravenzande, The Netherlands. None of the funding organizations had a role in the design or conduct of the research.
Footnotes
 Disclosure: T. Hillenaar, None; R.H.H. Cals, None; P.H.C. Eilers, None; R.J. Wubbels, None; H. van Cleynenbreugel, None; L. Remeijer, None
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Figure 1.
 
Backscatter measurements by IVCM. The IVCM images are 425 × 320 μm, with the bar representing 50 μm. Six characteristic layers of a normal human cornea and their position on the z-scan curve: (A) corneal endothelium; (B) corneal stroma showing a large straight nerve fiber; (C) the anterior stroma, characterized by a higher keratocyte density compared to middle and posterior thirds of the stroma. 10 ; (D) subbasal nerve plexus; (E) basal epithelial cell layer; and (F) superficial epithelial cells. (a–g) Seven variants of corneal backscatter. For variants a to e, mean backscatter per image was calculated in three subsequent passes through the cornea. 9 Variants f and g represent mean peak values of the three subsequent passes in one scan. Backscatter of the (a) cornea, (b) stroma, (c) posterior third of the stroma, (d) middle third of the stroma, (e) anterior third of the stroma and of the (f) SP and (g) EV.
Figure 1.
 
Backscatter measurements by IVCM. The IVCM images are 425 × 320 μm, with the bar representing 50 μm. Six characteristic layers of a normal human cornea and their position on the z-scan curve: (A) corneal endothelium; (B) corneal stroma showing a large straight nerve fiber; (C) the anterior stroma, characterized by a higher keratocyte density compared to middle and posterior thirds of the stroma. 10 ; (D) subbasal nerve plexus; (E) basal epithelial cell layer; and (F) superficial epithelial cells. (a–g) Seven variants of corneal backscatter. For variants a to e, mean backscatter per image was calculated in three subsequent passes through the cornea. 9 Variants f and g represent mean peak values of the three subsequent passes in one scan. Backscatter of the (a) cornea, (b) stroma, (c) posterior third of the stroma, (d) middle third of the stroma, (e) anterior third of the stroma and of the (f) SP and (g) EV.
Figure 2.
 
Age relatedness of corneal backscatter. (A) Mean corneal backscatter remained relatively stable with increasing age. Two outliers were seen at the higher end of the age spectrum (arrows). (B) Backscatter in the anterior third of the stroma per age category. After an initial decrease up to 50 years of age, backscatter in the anterior stroma showed an increase in the last two age categories. The last age category spanned 20 years instead of 10 years and was, for the purpose of this figure, divided into two decades, which comprised n = 42* and n = 18** eyes. Error bars represent 95% confidence intervals. (C) CCT correlated negatively with mean corneal backscatter (regression coefficient = −1.21 ± 0.25, P < 0.0001). (D) IOP did not correlate with mean corneal backscatter.
Figure 2.
 
Age relatedness of corneal backscatter. (A) Mean corneal backscatter remained relatively stable with increasing age. Two outliers were seen at the higher end of the age spectrum (arrows). (B) Backscatter in the anterior third of the stroma per age category. After an initial decrease up to 50 years of age, backscatter in the anterior stroma showed an increase in the last two age categories. The last age category spanned 20 years instead of 10 years and was, for the purpose of this figure, divided into two decades, which comprised n = 42* and n = 18** eyes. Error bars represent 95% confidence intervals. (C) CCT correlated negatively with mean corneal backscatter (regression coefficient = −1.21 ± 0.25, P < 0.0001). (D) IOP did not correlate with mean corneal backscatter.
Figure 3.
 
Diurnal variation of corneal backscatter. (A) Mean corneal backscatter varied significantly during the day: maximum variation 2.7%, P = 0.002. (B) CCT remained stable during the day. (C) Diurnal variation in IOP was exactly opposite to the change in corneal backscatter; maximum variation of IOP was 14.1%, P < 0.0001. Error bars represent 95% confidence intervals of the mean.
Figure 3.
 
Diurnal variation of corneal backscatter. (A) Mean corneal backscatter varied significantly during the day: maximum variation 2.7%, P = 0.002. (B) CCT remained stable during the day. (C) Diurnal variation in IOP was exactly opposite to the change in corneal backscatter; maximum variation of IOP was 14.1%, P < 0.0001. Error bars represent 95% confidence intervals of the mean.
Figure 4.
 
Future applications of corneal backscatter measurement. Images are 425 × 320 μm, with the bar representing 50 μm. (A, B) The EV can be used to objectively assess corneal hydration. 31 (A) The EV was 3792 SU in a patient with Fuchs endothelial dystrophy before DSAEK. (B) One month after DSAEK, corneal edema had diminished in the same patient: 1170 SU. (C, D) Subepithelial fibrosis may be assessed with the SP. (C) Characteristic reticular meshwork of subepithelial fibrosis in Fuchs endothelial dystrophy. (D) Exactly the same location was imaged in the same patient 6 months after DSAEK. The shape of the reticular meshwork had remained completely stable. Nevertheless, the SP declined from 5268 to 1984 SU. (E, F) Backscatter of the anterior, middle, and posterior parts of the stroma and their overall mean, may be used to monitor the inflammatory process in herpetic stromal keratitis. (E) During a recurrence, the stroma of this patient with herpes simplex keratitis showed an increase in backscatter: 3189 SU. (F) Mean stromal backscatter had returned to normal in the same patient 1 year after recurrence of the immune stromal keratitis: 1226 SU. All images (AF) were acquired with fixed light intensity of 72%, after calibration of backscatter analysis.
Figure 4.
 
Future applications of corneal backscatter measurement. Images are 425 × 320 μm, with the bar representing 50 μm. (A, B) The EV can be used to objectively assess corneal hydration. 31 (A) The EV was 3792 SU in a patient with Fuchs endothelial dystrophy before DSAEK. (B) One month after DSAEK, corneal edema had diminished in the same patient: 1170 SU. (C, D) Subepithelial fibrosis may be assessed with the SP. (C) Characteristic reticular meshwork of subepithelial fibrosis in Fuchs endothelial dystrophy. (D) Exactly the same location was imaged in the same patient 6 months after DSAEK. The shape of the reticular meshwork had remained completely stable. Nevertheless, the SP declined from 5268 to 1984 SU. (E, F) Backscatter of the anterior, middle, and posterior parts of the stroma and their overall mean, may be used to monitor the inflammatory process in herpetic stromal keratitis. (E) During a recurrence, the stroma of this patient with herpes simplex keratitis showed an increase in backscatter: 3189 SU. (F) Mean stromal backscatter had returned to normal in the same patient 1 year after recurrence of the immune stromal keratitis: 1226 SU. All images (AF) were acquired with fixed light intensity of 72%, after calibration of backscatter analysis.
Table 1.
 
Group Characteristics
Table 1.
 
Group Characteristics
Group 1 Sex, Age Relatedness Group 2 Diurnal Variation Group 3 Intersession Repeatability
Subjects, n 150 20 25
Mean age, y 45 (20–79) 38 (20–59) 44 (27–67)
Men, n 75 7 8
Eyes, n 300 40 50
Sessions, n 1 4 4
Time 9:00–12:00 9:00, 12:00, 15:00, 18:00 9:00–12:00
Month 0 0 0, 3, 6, 9
IVCM + + +
US pachymetry + +
GAT IOP + +
Table 2.
 
Sex Relatedness of Corneal Backscatter Measured by IVCM
Table 2.
 
Sex Relatedness of Corneal Backscatter Measured by IVCM
Men Women ΔSEX (%) P
Cornea 1248 ± 149 1206 ± 96 3.5 0.003
Stroma 1106 ± 181 1057 ± 108 4.6 0.005
Anterior stroma 1145 ± 177 1117 ± 125 2.5 NS
Mid stroma 962 ± 171 925 ± 106 4.1 0.02
Posterior stroma 1211 ± 232 1130 ± 135 7.2 0.001
Epithelial valley 1044 ± 103 1024 ± 99 1.9 NS
Subepithelial peak 1579 ± 252 1446 ± 177 9.2 0.0002
Table 3.
 
Age-Related Changes of Corneal Backscatter Measured by IVCM
Table 3.
 
Age-Related Changes of Corneal Backscatter Measured by IVCM
20–29 y 30–39 y 40–49 y 50–59 y 60–79 y Max ΔAGE (%) P
Cornea 1245 ± 100 1217 ± 97 1187 ± 106 1204 ± 113 1257 ± 123 5.9 NS
Stroma 1091 ± 114 1068 ± 118 1039 ± 119 1066 ± 125 1114 ± 144 7.2 0.05
Anterior stroma 1103 ± 110 1101 ± 131 1097 ± 146 1143 ± 135 1190 ± 148 8.5 0.0003
Mid stroma 958 ± 115 939 ± 118 903 ± 110 928 ± 115 960 ± 139 6.3 NS
Posterior stroma 1212 ± 162 1165 ± 143 1117 ± 143 1126 ± 172 1193 ± 180 8.5 NS
Epithelial valley 1085 ± 119 1017 ± 97 1008 ± 91 1014 ± 77 1039 ± 85 7.6 NS
Subepithelial peak 1520 ± 221 1459 ± 166 1457 ± 227 1545 ± 191 1560 ± 254 7.1 NS
Table 4.
 
Diurnal Variation of Corneal Backscatter Measured by IVCM
Table 4.
 
Diurnal Variation of Corneal Backscatter Measured by IVCM
9 h 12 h 15 h 18 h Max ΔDAY (%) P
Cornea 1220 ± 106 1238 ± 108 1253 ± 111 1250 ± 102 2.7 0.002
Stroma 1058 ± 119 1073 ± 119 1088 ± 120 1088 ± 115 2.8 0.0001
Anterior stroma 1085 ± 146 1108 ± 152 1122 ± 149 1114 ± 143 3.4 0.001
Mid stroma 924 ± 106 933 ± 101 948 ± 107 949 ± 103 2.7 0.0004
Posterior stroma 1165 ± 141 1179 ± 148 1194 ± 145 1200 ± 139 3.0 0.001
Epithelial valley 1053 ± 103 1081 ± 124 1087 ± 112 1092 ± 105 3.7 0.003
Subepithelial peak 1480 ± 211 1508 ± 218 1540 ± 231 1525 ± 210 4.1 0.01
Table 5.
 
Repeatability of Backscatter Measured by IVCM
Table 5.
 
Repeatability of Backscatter Measured by IVCM
Intrasession Intersession COVT (%)
COV (%) COR COV (%) COR
Cornea 4.2 143 3.2 111 5.3
Stroma 3.8 114 3.2 98 5.0
Anterior stroma 4.0 127 3.5 111 5.3
Mid stroma 3.7 98 3.4 88 5.0
Posterior stroma 5.2 170 4.3 140 6.8
Epithelial valley 3.8 111 3.0 84 4.8
Subepithelial peak 4.0 169 3.6 155 5.4
Table 6.
 
Generalized Normal Range and MDC95% of Corneal Backscatter
Table 6.
 
Generalized Normal Range and MDC95% of Corneal Backscatter
Mean 99% Reference Interval MDC95% (%)
Cornea 1222 966–1534 16
Stroma 1075 796–1435 15
Anterior stroma 1126 816–1531 16
Mid stroma 937 673–1285 15
Posterior stroma 1162 811–1635 20
Epithelial valley 1032 802–1316 15
Subepithelial peak 1508 1033–2156 16
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