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From the Tennent Institute of Ophthalmology, University of Glasgow, Glasgow, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. The performance of the system was first investigated by scanning a test object containing an air gap of known size. Measurements were repeated with water or glycerin in the gap. In the clinical setting, macular thickness measurements were obtained from a control group of 20 normal subjects. For analysis, these scans were divided into eight sections, each containing 10 A-scans.
RESULTS. The average gap thickness was found to be close to the true value in all cases. The overall coefficients of intersession reproducibility were less than 1% for the test object and 1.51% for the control group. There was no significant difference between scans acquired during different sessions. The overall coefficients of repeatability for the test object were between 0.2% and 1.1% and between 1% and 2% for the control group. The range of normal retinal thickness in terms of the 5th and 95th percentiles was 222 to 248 µm in women and 234 to 257 µm in men.
CONCLUSIONS. Measurements made from OCT scans were found to be accurate and precise. Introducing water or glycerin into the test object resulted in considerable degradation of the signal, but measurements of gap thickness were still shown to be accurate, precise, reproducible, and repeatable. Retinal thickness measurements in the macular area were repeatable and reproducible. This demonstrates that OCT is a useful tool in the monitoring of patients with conditions that affect macular thickness, even when there is considerable degradation of the OCT signal.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate changes in macular thickness it is first necessary to determine the range of retinal thickness in the normal population and to quantify the accuracy, reproducibility and repeatability of measurements made by the system. At present, there is only one commercially available OCT system, manufactured by Humphrey-Zeiss Medical Systems (San Leandro, CA), and several groups have evaluated the repeatability or reproducibility of both this prototype system and the commercially available one and have published values for the range of retinal thickness or NFL thickness in a control population.14 16 26 27 28 29 30 31 However, there do not seem to be consistent definitions of the terms repeatability and reproducibility. One purpose of this study was therefore to quantify both the repeatability and the reproducibility of the commercially available OCT scanner by basing our definitions of repeatability and reproducibility on the standards set by the British Standards Institution and the International Standards Organization,32 33 as recommended by Bland and Altman.34 In addition to assessing repeatability and reproducibility, it is important to quantify the accuracy and precision of the measurements made by the system, a concern that does not appear to have been investigated so far. It is also worth assessing the performance of the equipment in situations in which the reflected OCT signal is somewhat degraded and boundaries are less well defined. Thus, another purpose of this study was to analyze the performance of the system by scanning a custom-built object of precisely known size under three different conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The custom-built test object consisted of two 1-cm-thick plates of glass separated by four 200-µm-thick spacers. The thickness of the spacers was known to an accuracy of 0.5 µm. A technique called optical contacting made it possible to attach the spacers to the glass plates without the use of any sort of adhesive. This technique is a process by which two surfaces adhere to one another through molecular attraction. The surfaces of the plates to be contacted are parallel to within 0.5 arc-second, extremely flat, and cleaned to an exceptionally high degree. When brought together, the surfaces then adhere with no adhesive. This technique ensures that the thickness of the gap corresponds exactly to the thickness of the spacers. Thus, this object basically provided us with a gap of precisely known thickness. This gap could also be filled with liquids. Scans of the test object resulted in two reflecting bands representing the glass–air or glass–liquid boundaries. The plates of the test object were made of fused silica with a refractive index of 1.452 at 840 nm. Thus, imaging of the air-filled gap resulted in very strong reflections due to the large change in refractive index at the boundary. In the eye, reflections tend to be less pronounced; and to model the in vivo situation more effectively the experiment was therefore repeated with the gap filled with water (refractive index, 1.333) and glycerin (refractive index, 1.473). This had the effect of degrading the intensity of the reflected signal and making the glass–liquid boundary far less well defined.
The OCT software assumes a refractive index of 1.38 for retinal tissue (this index value was provided by the manufacturers of the system); thus, measurements made from the A-scan were multiplied by this index to convert them back into measurements in air. These values were then divided by the refractive index of the material within the gap to arrive at the true gap thickness, as measured from the OCT scan. The results were then compared with the known thickness of the gap.
Subjects
Twenty volunteers (10 men and 10 women), ranging in age from 21
to 57 years (average age, 31.9 years) participated in this study. The
study was conducted according to the tenets of the Declaration of
Helsinki, and volunteers gave informed consent after the nature and
intent of the study had been fully explained to them. The exclusion
criterion was history of known retinal disease. All scanning was
performed in the right eye, which was dilated with 1% tropicamide.
Scanning
The known distance between the plates of the test object assumes
an incident beam normal to the sample. Thus tilting the sample would
increase the distance traveled by the OCT beam, leading to a higher
measurement. Each pixel within the A-scan represents a distance of 4
µm in retinal tissue that corresponds to 5.5 µm in air, 4.1 µm in
water and 3.7 µm in glycerin; thus, OCT measurements can distinguish
only between measurements that vary by more than these amounts. From
geometric calculations and experimentation, it was found that tilts
within 10° from normal caused inaccuracies that were less than the
intrinsic thickness resolution of the system. Thus, the positioning of
the test object was not particularly critical for making accurate
measurements. It was observed that the intensity of the reflections
from the interfaces varied with different focusing and polarization
settings. In the case of the air-filled gap, these were adjusted to
give the strongest possible signal. Several scans across the surface of
the object were then acquired.
For the cases in which the gap was filled with liquid, we wanted to quantify the degradation of the signal. The object was first set up with an air-filled gap, as just explained, and then the liquid was introduced carefully without altering the position of the object or the polarization and focusing settings. Scans were acquired before and after the liquid was inserted, so that the reduction in intensity of the reflections could be assessed.
In our study of normal subjects we initially took a series of horizontal single-line scans across the fovea in each subject. Scans were repositioned, using the repeat-scan feature that provides a landmark cursor to facilitate the repeat positioning of subsequent scans. We then discarded any scans in which the landmark cursor was not in the correct position, thus ensuring that all the saved scans were of exactly the same portion of retina. However, after careful analysis of the fundus pictures it was discovered that there was a degree of inaccuracy in the positioning of this landmark cursor. We found that there was some displacement, even between scans in which the landmark cursor appeared to be in exactly the same position. This displacement was of the order of 0.2 to 0.3 mm. In the region of the fovea where the thickness of the retina is varying, a shift of this amount could cause considerable variations in the measured retinal thickness and would lead to inaccuracies in the coefficients of repeatability and reproducibility.
In an attempt to minimize the effects of landmark positioning errors, we therefore decided to scan across a band rather than across a single line. This was achieved by having a number of very closely spaced scan lines using the raster six-lines option, which allows six tomographic scans to be acquired in succession. In this scanning mode, an aiming rectangle of adjustable dimensions is displayed on the fundus-viewing unit. The width of this rectangle determines the length of the scan lines, whereas its height determines the spacing between the scans. In this case, the width of the aiming rectangle was set at 4 mm and its height was 0.5 mm. Thus, the spacing between successive scans was 0.1 mm. The aiming rectangle was positioned such that at least four of the scans traversed and were centered on the foveal pit. These four scans therefore covered a vertical length of 0.3 mm, which corresponds to the maximum error in positioning found from our initial investigation. These four scans from each group were then used in subsequent calculations. The focusing and polarization settings were adjusted so that the best-quality image was obtained.
Our definitions of repeatability and reproducibility were based on the definitions adopted by the British Standards Institution.32 33 Under repeatability conditions, independent test results are obtained with the same method on the same subject by the same operator and on the same set of equipment, with the shortest time lapse possible between successive sets of readings. We investigated repeatability initially on the test object by acquiring 10 scans in rapid succession. Repeatability on the control group was investigated by obtaining 10 sets of six tomographic scans from the same subject. All scanning was performed by the same operator. The time elapsed between successive sets of scans corresponded to the time taken to set up and position the aiming rectangle for a new set of tomographic scans and was always less than 1 minute. Repeatability was investigated for three different subjects.
Under reproducibility conditions sets of readings are obtained using the same method but on different occasions. Intersession reproducibility for the test object was investigated by acquiring readings in the morning and afternoon on five consecutive days. The time separation between the morning and afternoon sessions was at least 5 hours, and the OCT scanner was not switched off during this period. The temperature of the room varied by approximately 2° during that time. We then analyzed intersession reproducibility in the clinical setting for each of the 20 subjects by obtaining two sets of six tomographic scans with a minimum time separation of 30 minutes.
Analysis
A-scans of the test object showed two peaks corresponding to the
edges of the air gap. We decided to measure the thickness of the gap by
considering the distance between the two maxima. Computer programs that
identified the peaks and calculated the distance between them were
developed, because the software provided with the OCT scanner could not
perform these functions. When the gap was filled with air, the gap
edges were very well defined, and the two maxima corresponding to the
glass–air interfaces were easily identifiable on each of the A-scans.
Filling the gap with either water or glycerin caused a reduction in the
overall intensity of the reflections from the interfaces. At some
positions along the scan line, the returning reflection was so weak
that it fell below the noise threshold, and this meant that the gap
edges no longer appeared as continuous lines on the B-scan but had a
more patchy appearance. At these positions, it was impossible to
identify the two maxima corresponding to the gap edges from the A-scan.
Thus, thickness measurements were made only from the A-scans in which
the two maxima could clearly be identified.
To quantify the degradation of the signal caused by introducing a liquid into the air gap we calculated the percentage reduction in reflectance. For each B-scan acquired as part of the repeatability study, we selected the A-scans in which the two maxima were clearly identifiable and quantified the intensity of the reflections from the interfaces These values were then averaged over the entire B-scan. This value was then divided by the average intensity calculated from the scans acquired just before the liquid was introduced. In the water-filled condition, the intensity of the reflection from anterior edge of the gap (closest to the machine head) was found to be 48.5% of the air-filled condition; for the posterior edge it was 57.0%. With glycerin in the gap, these values were 42.9% for the anterior edge and 47.7% for the posterior.
For the water-filled gap, an average of 97 A-scans per B-scan showed two easily identifiable maxima, whereas in the glycerin-filled condition this value was 45 A-scans per B-scan.
In the normal control study, four scans that traversed the foveal pit were selected from each set of six tomographic scans, and only these were used for determining the coefficients of repeatability and reproducibility. These scans were labeled level 1 to level 4, with level 1 being the most inferior scan of the set. The retinal thickness along each point of each scan was found by using the retinal thickness tool provided with the OCT software. This software assumes that the first highly reflecting band corresponds to vitreoretinal interface and that the second corresponds to the retinal pigment epithelium. Thus, retinal thickness measurements are made by evaluating the displacement between the anterior surfaces of these two interfaces. The results from this tool cannot be exported directly from the system; however, the manufacturer provides a separate program that exports this data as a text file. Thickness values were thus exported for each scan, and any obvious errors in boundary detection were corrected manually.
The center of each scan was taken to be the thinnest point of the retina, which was assumed to correspond to the deepest portion of the foveal pit. The A-scan at the center of the scan was labeled A0. Scans to the left of this were labeled A-1 to A-49, and scans to the right were labeled A1 to A50. Each scan was then divided into eight sections, each containing 10 A-scans. Sections to the left of the center were labeled S-1 to S-4 and those to the right were labeled S1 to S4 as shown in Figure 1 . Thus, for each B-scan, only the 80 A-scans from A-39 to A40 were used in the calculations. The retinal thickness obtained from each of these 80 scans was averaged across the four levels in each set in an attempt to minimize the effects of errors in scan positioning. Thus, we were left with a single set of 80 thickness values from each group of tomographic scans. The overall average retinal thickness (the average of these 80 thickness values) and the average retinal thickness per section were calculated for each group of data.
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As suggested by Bland and Altman34 who based their definitions on the recommendations of the British Standards Institution, the coefficient of intersession reproducibility was defined as the SD of the differences between pairs of measurements obtained during different sessions divided by the average of the means of each pair of readings. Intersession repeatability was evaluated for the test object from the average air gap thickness computed from each session. The overall coefficient of intersession reproducibility for the control group was calculated from the 20 overall average retinal thickness values. Coefficients of reproducibility were also calculated for each of the eight retinal sections. A graph of differences against means was plotted both for the overall average retinal thickness and for each section. In both cases, the Wilcoxon matched-pairs test (5% significance level) was also used to establish whether there was any statistically significant difference between measurements obtained during different sessions.
The coefficient of repeatability obtained from the repeated administration of the test under identical conditions was defined as the SD of the difference from the mean of these repeat measurements divided by the average response. Coefficients of repeatability were calculated from the 10 consecutive scans of the test object, as well as in each of the three subjects participating in the repeatability study.
To establish whether scanning across a 0.3-mm band of retina, rather than across a single line, actually improved the repeatability, we also computed the coefficient of repeatability for each level and compared that with the value obtained from the average over four levels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intersession Reproducibility
Test Object.
The average gap thickness was calculated from the individual A-scan
thicknesses for each morning and afternoon session. These values were
then used to compute the overall coefficients of intersession
reproducibility, which were found to be 0.67% (air-filled), 1.05%
(water-filled), and 0.45% (glycerin-filled). The Wilcoxon
paired-measurement test (5% significance level) showed that there were
no statistically significant differences between the measurements
obtained in the morning and afternoon scanning sessions.
Control Group.
For each subject the overall average retinal thickness was calculated
for sessions 1 and 2. The overall coefficient of reproducibility was
then computed from these values and was found to be 1.51%. In
addition, we found the average retinal thickness for each of the eight
sections and computed the coefficient of intersession
reproducibility for each section. The results obtained are shown in
Table 1
. The intraclass correlation coefficient (ICC) for intersession
reproducibility was found to be 0.96. Graphs of differences against
means were plotted for the overall average retinal thickness values, as
well as for each section. We have shown only the graph of differences
against means for the overall retinal thickness (Fig. 2)
; the graphs for the individual sections were very similar in
appearance. In all cases it was found that 95% (19 of 20) of
differences fell within 2 SDs of the mean. According to the definitions
of the British Standards Institution33
this indicates
reproducibility in overall and sectional retinal thickness
measurements. The Wilcoxon paired measurement test (5% significance
level) was also performed on the overall retinal thickness values and
on the values for each retinal section. No statistically significant
differences were found between the two sets of data. There is therefore
no evidence to suggest that slight variations in room temperature (on
the order of 2°C) have any effect on the performance of the OCT
scanner. With a study involving 20 patients, the probability is 80%
that the study will detect a difference in morning and afternoon
sessions if the true difference between sessions is 2.18 µm
(two-tailed 5% significance level).
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Control Group.
For each of the three subjects in the repeatability study we computed
the overall coefficient of repeatability by analyzing the overall
average retinal thickness from each set of readings. We then computed
the coefficient of repeatability for each of the eight retinal
sections. The results obtained are shown in Table 2
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Retinal Thickness
The median and the 5th and 95th percentiles of the overall and
sectional retinal thickness values are shown in Table 3
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The overall coefficients of intersession reproducibility were found to be between 0.67% and 1.05% for the test object and 1.5% for the control group. Sectional coefficients of reproducibility were all less than 5%. This indicates that any significant variation in retinal thickness measurements from different scanning sessions is likely to be due to changes in the patient’s retinal thickness rather than to inconsistencies in the values given by the OCT system. Thus, OCT may be used to monitor patients with conditions that affect the thickness of the retina in the macular region, even in situations in which the retinal interfaces are poorly defined.
The average test object gap thicknesses computed under the three different conditions agree very closely with the known thickness of the air gap, which was 200 µm. This indicates a high level of accuracy and precision in the measurements made by the system, even in situations in which the OCT signal is relatively weak.
From Table 2 it is clear to see that the repeatability of the retinal measurements made over a band of 0.3 mm is consistently better than when repeated scans are made across a single line. This confirms that slight errors in scan positioning occur and consequently, that our method of acquiring a series of scans across a 0.3-mm band yields more reliable measures of repeatability and reproducibility of the system than simply scanning repeatedly across a single line. These errors in positioning may be partly because the quality of the fundus picture displayed on the fundus-viewing monitor is somewhat poor. Moreover, whereas in the slow-scanning mode, the landmark cursor tends to lose its definition, thus making it very difficult to ensure that it remains in the correct position. It is hoped that future versions of the hardware will include a better quality fundus-viewing unit that would enable more precise repeat scanning.
Our methods of quantifying repeatability and reproducibility of retinal thickness measurements in the foveal region differ slightly from those used in other publications; however, the results obtained are quite similar. Koozekanani et al.27 analyzed sets of scans obtained during independent measuring sessions. They found that there were no significant differences between different sessions or between different scans within the same session; however, they do not specify which statistical test was used. We performed the Wilcoxon matched-pairs test on the data obtained under reproducibility conditions and found that there were no significant differences between the sets of data acquired during different scanning sessions. This is true both for the overall retinal thickness as well as for the thickness in each retinal section.
Our method of subdividing each scan into sections containing 10 A-scans is very similar to the system adopted by Baumann et al.26 They divided their images into seven regions, each containing 10 A-scans, and computed the coefficient of variation for retinal thickness measurements made in each of these sections. They found that the greatest coefficient of variation occurred in the central section, which covered a retinal length of 320 µm centered on the foveal pit. Sections closest to the fixation point showed less reproducibility than those farther away. We calculated the coefficients of repeatability and reproducibility for each of our retinal sections and similarly found that these coefficients tended to be highest for regions S1 and S-1, which correspond to the sections closest to the center of the fovea. In this region the retinal thickness varies, and hence any errors in scan positioning will causes variations in the measured thickness of this region. We have attempted to compensate for this by scanning across a 0.3-mm band. However, although we have shown that this reduces the effects of errors in positioning, the higher coefficients of repeatability and reproducibility in these regions relative to other regions indicate that there is still a degree of inaccuracy in the positioning of scans.
It is also important to establish confidence intervals for retinal thickness in the normal population. We therefore computed the median and the 5th and 95th percentiles for retinal thickness in the macular region. Our control group was not ideal, because all our subjects were relatively young. Nevertheless, our results compare very well with those obtained by other investigators.
The size of our sample and the mean age are similar to those investigated by Baumann et al.,26 and our sectional results are comparable to those quoted in their publication. The average retinal thickness for sections S3 and S4 together was 271 ± 16 µm. This represents an average over an 0.8-mm section of retina at a distance of 0.8 mm from the foveal pit, and the result is almost identical with the average retinal thickness of 274 ± 17 µm of Koozekanani et al.27 for a 1-mm section at a distance of 0.75 mm from the foveal pit. The average retinal thickness for sections S1 and S-1 together was 178 ± 18 µm in the women and 190 ± 24 µm in the men. These values are higher than the foveal thicknesses of Hee et al.14 of 169 ± 4 µm for the female and 181 ± 4 µm for the male subjects. This difference is probably because Hee et al. analyzed a circular region of 500-µm diameter, whereas our analysis was performed on a larger rectangular region of 800 x 300 µm.
In this study we concentrated on total retinal thickness within the macular area and showed that measurements made in this area are accurate and precise and that they demonstrate a high level of repeatability and reproducibility. This implies that OCT can reliably be used to monitor patients with conditions that affect macular thickness. A simple model has shown that in patients in which interfaces are not very well defined, the measurements made nevertheless agree very closely with the known value of the distance being measured and that these measurements still show a high degree of repeatability and reproducibility. This has important implications for assessing changes in macular thickness in patients affected by conditions, such as macular edema, which may degrade the quality of the OCT image. In the assessment of conditions such as glaucoma and macular edema, it is becoming increasingly common to make use of six radial scans to create a retinal map. A possible extension of this study would be to assess the repeatability and reproducibility of the OCT scanner in this situation.
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Footnotes |
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Submitted for publication March 21, 2001; revised August 24, 2001; accepted September 10, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: David Keating, Electrodiagnostic Imaging Unit, Tennent Institute of Ophthalmology, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, United Kingdom; d.keating{at}clinmed.gla.ac.uk
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