Optometry & Vision Science:
Reproducibility-Repeatability of Choroidal Thickness Calculation Using Optical Coherence Tomography
Benavente-Pérez, Alexandra*; Hosking, Sarah L.†; Logan, Nicola S.‡; Bansal, Dheeraj§
†PhD, MCOptom, FAAO
Department of Biological Sciences, SUNY State College of Optometry, New York, New York (AB-P), Ophthalmic Research Group, School of Life and Health Sciences, Aston University, Birmingham, United Kingdom (SLH, NSL), Department of Optometry and Visual Science, City University, London, United Kingdom (SLH), Department of Ophthalmology, University of Melbourne, Melbourne, Victoria, Australia (SLH), and InnZ Medical AB, Ljungskile, Sweden (DB).
Received March 8, 2010; accepted June 22, 2010.
Alexandra Benavente-Pérez; Department of Biological Sciences; SUNY College of Optometry; 33 W 42nd Street; New York 10036, New York; e-mail: firstname.lastname@example.org
Purpose. To evaluate the repeatability and reproducibility of subfoveal choroidal thickness (CT) calculations performed manually using optical coherence tomography (OCT).
Methods. The CT was imaged in vivo at each of two visits on 11 healthy volunteers (mean age, 35.72 ± 13.19 years) using the spectral domain OCT. CT was manually measured after applying ImageJ processing filters on 15 radial subfoveal scans. Each radial scan was spaced 12° from each other and contained 2500 A-scans. The coefficient of variability, coefficient of repeatability (CoR), coefficient of reproducibility, and intraclass correlation coefficient determined the reproducibility and repeatability of the calculation. Axial length (AL) and mean spherical equivalent refractive error were measured with the IOLMaster and an open view autorefractor to study their potential relationship with CT.
Results. The within-visit and between-visit coefficient of variability, CoR, coefficient of reproducibility, and intraclass correlation coefficient were 0.80, 2.97% 2.44%, and 99%, respectively. The subfoveal CT correlated significantly with AL (R = −0.60, p = 0.05).
Conclusions. The subfoveal CT could be measured manually in vivo using OCT and the readings obtained from the healthy subjects evaluated were repeatable and reproducible. It is proposed that OCT could be a useful instrument to perform in vivo assessment and monitoring of CT changes in retinal disease. The preliminary results suggest a negative correlation between subfoveal CT and AL in such a way that it decreases with increasing AL but not with refractive error.
Until the recent application of optical coherence tomography (OCT) to the choroid,1,2 in vivo detection of the human choroid had mainly been perform using non-imaging techniques such as the high frequency ultrasound used in animals3–5 or partial coherence interferometry in humans.6 Brown et al. were able to describe diurnal fluctuations in choroidal thickness using partial coherence interferometry; however, they pointed out how the signal of the choroidal interface in some cases appeared attenuated, which did not facilitate the adequate identification of the choroid/retinal peaks in the human eye.6
OCT uses low-coherence interferometry and allows the identification of the retinal structures, whose measurement correlate well with the morphology of the foveal and parafoveal retinal area.2,7,8 Following the topographical study of the retinal and choroidal measurements in healthy subjects,1,7,8 the interest shifted toward the analysis of the morphological changes that occur in choroidal-related degeneration.9–11 Ikuno and Tano9 reported the clinical presence of choroidal thinning at the fovea of highly myopic patients but such correlation did not appear significant when healthy Japanese subjects with myopia smaller than 6.00 D where evaluated.10 Following the same line of investigation, Fujiwara et al.11 assessed highly myopic eyes and found a negative relationship between CT and refractive error, and from the same research group, a later article was published suggesting that the choroids of patients with central serous retinopathy are significantly thicker.12
However, to date, the repeatability and reproducibility of the subfoveal CT calculations has not been evaluated in vivo in a cohort of healthy Caucasian eyes. From the theoretical perspective, this study furthers our insight in the physiological biometry of the choroid and the role the OCT may play in the investigation of retinal disease as a potential diagnostic tool to perform in vivo assessment and monitoring of CT changes in the human eye, known to relate to pathological and vision-threatening conditions.9–12 The aim of this study was to assess the repeatability and reproducibility of the subfoveal CT calculations performed manually using Spectral OCT Copernicus (SOCT) in healthy human eyes of Caucasian origin.
Eleven healthy volunteers recruited from the students and staff at Aston University, (Birmingham, UK) participated in the study (Table 1). One eye of each participant was randomly selected. All the participants were healthy volunteers and this was confirmed by ophthalmologic investigation of the fundus, blood pressure measurements, and detailed recording of systemic and ocular history and symptoms. Exclusion factors included any ocular or systemic disorders or interventions.
Aston University Ethics Committee approved the study and the study adhered to the tenets of the declaration of Helsinki. Written and verbal information was given to the volunteers before data collection and all the participants gave informed written consent.
Choroidal Thickness Measurements
The choroid was imaged using the SOCT Copernicus (Optopol SA, Poland), designed and built at the Physics Institute of University of Nicolaus Copernicus in Torun, Poland.7,8 The SOCT is non-invasive and uses a light source with a wavelength of 830 nm. The interference signal from the interferometer is detected by a spectrometer (reflecting holographic grating with 1800 lines per mm), which provides a transversal resolution of 6 μm and a tangential resolution of 10 μm with an examination speed of 26,000 A-scans per second. The maximum number of A-scans per B scan is 7,000, and all head movements are automated motorized and controlled from a computer screen.
Axial length (AL) was measured using the IOLMaster (Carl Zeiss, Jena, Germany). Objective non-cycloplegic refraction was obtained using the Shin-Nippon SRW 5000 Autorefractor (Tokyo, Japan) and a high contrast Maltese cross at 6 m as a fixation target.
The data were collected on two consecutive days to evaluate the within-visit and between-visit reproducibility. Because diurnal variations in CT have been reported in animal and human studies,6,13 the two visits were performed at approximately the same time on consecutive days. Twelve subfoveal radial scans were taken per patient per day, from which 10 were chosen based on best image quality. Based on previous OCT studies assessing the repeatability of the retinal thickness measurement,14,15 the scan of choice was a radial scan comprising 15 radial line scans of 7 mm in length arranged in a star-like pattern centered on the fovea (Fig. 1). Each radial scan was spaced 12° from each other and contained 2500 A-scans. The scanning time of the SD OCT Copernicus varies from 0.01 s if the scan of choice is a 2-D scan (512 A-scans), to 1.2 s if the scan protocol is a 3-D scan (50 B-scans with 800 A-scans). In this study, the scanning time was 0.6 s per scan and only the subfoveal choroid was measured (Fig. 2).
The CT calculations were performed manually after the image was filtered with ImageJ to ensure adequate positioning of the calipers provided by the software of the instrument. The calculations were made by the same observer, who was blind to the patient's clinical record. In detail, the background was subtracted (image 2, top left) and sharpened (image 2, top right), and then converted to a binary image (image 2, bottom left) where the edges could be detected (image 2, bottom right). The foveal area was first delimited using a horizontal marker (Fig. 3, line 1) that was later used as a reference to adjust a perpendicular line through the center of the foveal pit (Fig. 3, line 2). The choroid was measured from the outer edge of the retinal pigment epithelium to the scleral boundary as defined by edge detection to the furthest point of the large choroidal vessels (gray arrow).
The CT readings obtained were tested for normality using the Shapiro-Wilk test before further statistical analyses. For each subfoveal scan, the coefficient of repeatability (CoR) and coefficient of variation (CoV) (both expressed as a percentage) were determined based on the recommendations of the British Standard Institution. Additionally, the intraclass correlation coefficient (ICC) was calculated. Pearson product-moment correlation (R) was used to evaluate the relationship between CT, AL, and mean spherical equivalent refractive error.
Power statistics using data from previous articles1,10 revealed that a sample size with a statistical power of 80%, allocation ratio 1:1 and a significant value of 5% (alpha = 0.05) required a sample of 10 participants to perform a normative evaluation of the data (sample size calculations were performed following Simple Interactive Statistical Analysis, SISA).
Ten radial scans comprising 12 radial line scans were taken for each participant. The CT for each radial location and total CT are given in Table 2. The Shapiro-Wilk test showed a p value equal to p = 0.30, which indicated that the measurements of CT were normally distributed.
The CoV, CoR, coefficient of reproducibility (CoRepro), and ICC for the mean of the subfoveal CT measurements were the following: CoV 0.80%, CoR 2.97%, CoRepro 2.44%, and ICC 99%. A Bland Altman difference against mean plot was used to represent the mean CT differences for each participant between visits as a function of the mean CT (Fig. 4). CT was found to correlate negatively with AL (R = −0.60, p = 0.05) (Fig. 5) but not with refractive error (r = 0.31, p > 0.5).
This study evaluated the feasibility of using the SOCT to determine subfoveal CT in vivo. The coefficients of variability, repeatability, and reproducibility obtained in this study were comparable to those obtained in studies where the retinal thickness was measured, which indicates that the SOCT may be used for the calculation of CT in vivo. Additionally, in the small sample of participants evaluated, the thickness of the subfoveal choroid was found to correlate significantly with AL.
Six macular radial scans (based on previous OCT studies assessing the repeatability of the retinal thickness measurement14,15) were taken per participant, each of which in turn comprised 15 radial line scans. This resulted in a total of 90 images analyzed per subject. The radial scans were 7 mm in length arranged in a star-like pattern with the center on the fovea and spaced 12° from each other. The coefficients of variability, repeatability, and reproducibility were 0.80%, 2.97%, and 2.44% respectively, with an ICC of 99%, which were comparable with past retinal articles that reported good between session and within-session indices of reproducibility and repeatability (CoR: 0.89% to 1.51%, CoRepro 1% to 6%).14,15 However, the interest in the measurement of CT is so recent that limited data is available from healthy subjects to allow comparison with this study. The average subfoveal CT described in past articles ranged from 287 ± 76 (no ethnicity specified)1 to 354 ± 111 μm (Japanese subjects)10 and used EDI OCT and high penetration OCT, respectively. This study describes an average estimation of subfoveal CT of 448.48 ± 81.99 μm in younger healthy Caucasians (20 to 49 years old), which is hypothesized to be due to the negative relationship reported between age and subfoveal CT in healthy subjects.1,10 Because of the limited number of publications available on normative data for the measurement of subfoveal CT, this study also used a Bland-Altman plot to provide an additional qualitative way of analyzing the between-visit reproducibility of the instrument. None of the measurements fell outside the upper and lower limits in the graph after assessing the width of the 95% confidence interval, which agrees with the good coefficients previously discussed.
The results of the present article additionally describe a significant correlation between subfoveal CT and AL such that macular CT decreased with increasing AL. However, caution should be exercised when interpreting the findings because of the small sample size evaluated. AL is known as an important structural correlate in eye growth and myopia16–22 and one of the predictive factors, among others such as age and ethnicity, of the retinal nerve fiber layer thickness measured using OCT.23,24 Both children and adults exhibit significantly thinner macula with increasing AL,25–27 and AL has also very recently been found to correlate with the height of the posterior staphyloma in highly myopic patients.9 The present study was limited by the low number of subjects, the relatively low range of prescriptions included as well as the manual nature of the calculations. Consequently, a larger study is necessary to verify and validate our observation.
From a clinical perspective, it is important to highlight the increasing role that the OCT might play in the diagnosis and management of retinal disease in future practice28 because of its ability to quantify CT in vivo and non-invasively.12
In summary, the coefficients of variability, repeatability, and reproducibility obtained in this pilot study, suggest that it is feasible to measure the subfoveal CT in vivo manually. Future advancements with automated calculations of CT would improve the technique and would facilitate the possibility to generate studies where the choroidal biometry in human eyes could be monitored. It is, therefore, proposed that OCT could be a useful instrument to perform in vivo assessment and monitoring of CT changes with eye growth and retinal disease in the human eye.
We thank William Bourassa Jr, for his invaluable graphics assistance.
Department of Biological Sciences
SUNY College of Optometry
33 W 42nd Street
New York 10036, New York
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