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Arquivos Brasileiros de Oftalmologia

Print version ISSN 0004-2749On-line version ISSN 1678-2925

Arq. Bras. Oftalmol. vol.80 no.6 São Paulo Nov./Dec. 2017 

Original Articles

Repeatability and reproducibility of automatic segmentation of retinal layers in healthy subjects using Spectralis optical coherence tomography

Repetibilidade e reprodutibilidade da segmentação automática de camadas retinianas em sujeitos saudáveis com tomografia de coerência óptica Spectralis

Ersan Çetinkaya1 

Rahmi Duman2 

Reşat Duman2 

Mehmet Cem Sabaner2 

1Department of Ophthalmology, Manisa State Hospital, Manisa, Turkey.

2Department of Ophthalmology, School of Medicine, Afyon Kocatepe University, Afyonkarahisar, Turkey.



To evaluate the repeatability and reproducibility of automatic segmentation in healthy subjects using a Spectralis optical coherence tomography (OCT) system.


A total of 60 eyes from 60 patients were included in this prospective study. Spectral-domain optical coherence tomography images were generated using the Spectralis OCT system. An automated algorithm was used to segment the macular retina into nine layers and evaluate the thickness of each layer in the foveal, inner, and outer Early Treatment Diabetic Retinopathy Study (ETDRS) subfield rings. The eyes were imaged three times by an examiner to assess intraobserver repeatability and imaged once by a second examiner to assess interobserver reproducibility. The first scan was used for reference, whereas the second and third scans were collected using the device’s follow-up mode. Intraclass correlation coefficients (ICCs) of repeatability and reproducibility were analyzed.


The examiners achieved high repeatability and reproducibility for all parameters. Good agreement was found for all parameters in all ETDRS subdivisions with an ICC of >0.96 for all measurements.


It is possible to obtain cross-sections from the same location using the device’s follow-up mode, making it virtually impossible to distinguish between repeated measurements taken with the device by different examiners.

Keywords: Retina; Macula lutea; Tomography, optical coherence; Reproducibility of results; Image processing, Computer-assisted



Avaliar a repetibilidade e a reprodutibilidade da segmentação automática com tomografia de coerência óptica Spectralis em indivíduos saudáveis.


Foram incluídos neste estudo prospectivo um total de 60 olhos de 60 pacientes. As imagens de tomografia de coerência óptica de domínio espectral (SD-OCT) foram geradas com Spectralis OCT (Heidelberg Engineering, Heidelberg, Alemanha). Um algoritmo automatizado foi utilizado para segmentar a retina macular em nove camadas e quantificar a espessura de cada camada em anéis de subcampo ETDRS fovetais, internos e externos. Os olhos de cada paciente foram imaginados três vezes pelo primeiro examinador para avaliar a repetibilidade intra-observador e uma vez pelo segundo examinador para avaliar a reprodutibilidade entre observadores. A primeira verificação foi definida como a varredura de referência, enquanto a segunda e a terceira varredura foram as varreduras de seguimento e foram realizadas com o uso do modo de acompanhamento, respectivamente. O coeficiente de correlação intraclasse (ICC) de repetibilidade e reprodutibilidade foi analisado. Repetibilidade e reprodutibilidade das medidas obtidas foram analisadas utilizando o coeficiente de correlação intraclasse.


Os examinadores alcançaram alta repetibilidade e reprodutibilidade para todos os parâmetros. Foi encontrado um bom acordo para todos os parâmetros em todas as subdivisões de ETDRS com um coeficientes de correlação intraclasse superior a 0,96 para todas as medições.


É possível obter seções transversais do mesmo local usando o modo de acompanhamento, o que torna praticamente impossível distinguir entre medições repetidas do dispositivo independentemente do examinador.

Descritores: Retina; Mácula lútea; Tomografia de coerência óptica; Reprodutibilidade dos testes; Processamento de imagem assistida por computador


Optical coherence tomography (OCT) is a well-known tool for the high-resolution assessment of retinal pathologies. Spectral-domain OCT (SD-OCT) is a more recent technique that allows the imaging of ocular structures with faster scan rates than OCT(1-2). The evaluation of intraretinal layer thickness is becoming increasingly important for the diagnosis and monitoring of various ocular diseases(3). However, few studies have assessed the repeatability and reproducibility of automated total retinal thickness measurements using different SD-OCT instruments in healthy individuals(4,5) or those with ocular pathology(6-11).

The aim of this study was to investigate the repeatability and reproducibility of automatic segmentation of nine intraretinal layers in healthy subjects using a SD-OCT system.



This study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Afyon Kocatepe University. Healthy subjects (28 males, 32 females) who were admitted to the ophthalmology department for regular ophthalmological examinations with no history of ocular disease except for refractive errors were included, and each signed an informed consent form. The exclusion criteria were ocular surface disorder, corneal disease, previous corneal surgery, contact lens wear, history of ocular trauma or inflammation, and systemic disease.

Measurement device

Retinal OCT imaging was performed using the Spectralis OCT system (Heidelberg Engineering GmbH, Heidelberg, Germany). Segmentation of the retinal layers from each SD-OCT scan was performed using the built-in Spectralis mapping software the Heidelberg Eye Explorer (version 6.0). Spectralis segmentation software was used to obtain the following thickness measurements: total retinal thickness (Retina), retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and retinal pigment epithelium (RPE). The automatic segmentation tool of the posterior pole scan also provides the thickness of the inner retinal layer (IRL), measured from the internal limiting membrane to the external limiting membrane (ELM), and that of the outer retinal layer (ORL), measured from the ELM to the Bruch membrane (Figure 1). The Spectralis mapping software generates automated retinal thickness measurements based on analyses of the central, inner ring, and outer ring subfields as defined by the Early Treatment Diabetic Retinopathy Study (ETDRS)(12).

Figure 1 Segmented view of the retinal layers created using the Heidelberg Spectralis automatic segmentation analysis program. 

Measurement technique

Measurements were taken according to the guidelines of the respective device manufacturers. Before imaging, all eyes were subjected to an ocular examination including visual acuity testing, auto-refraction, intraocular pressure, and ophthalmoscopic examination. After enrollment, all eyes were imaged without mydriasis. Three repeated measurements were performed within a short period of time on the same day by a single examiner (EC) to test the intraobserver repeatability. The first examination was marked as the reference in the follow-up mode, a special feature of the Heidelberg SD-OCT system that allows scanning of the same part of the retina during follow-up visits. SD-OCT imaging was also performed once by another examiner (MCS) on the same day to test interobserver reproducibility. The thicknesses of all layers within the central ETDRS zones of 0-1000 and 1000-3000 µm (inner ring) and 3000-6000 µm diameter (outer ring) were recorded for each scan (Figure 2).

Figure 2 1: central area; 2-5: inner ring: 6-9, outer ring. 

Statistical analysis

All of the statistical analyses were performed by Statistical Package for the Social Sciences software (version 17.0 for Windows; SPSS Inc., Chicago, IL, USA). Descriptive statistics are shown as mean ± standard deviation. The intraobserver repeatability was measured with three OCT images obtained by the same operator, while the interobserver reproducibility was measured with two OCT images obtained by two different operators. The overall mean thickness and intraclass correlation coefficients (ICCs) were calculated to evaluate the repeatability and reproducibility of the thickness measurements. The overall mean thickness of the nine intraretinal layers along the central macular 6-mm scan length was determined as the mean of the three measurements by the same examiner. The paired ICCs were analyzed to evaluate the agreement of the thickness measurements between the two SD-OCT instruments.


Sixty volunteers (28 men, 32 women) aged 20-40 years (mean age, 30.26 ± 6.31 years) participated in this study. Table 1 shows the repeatability and reproducibility of the thickness measurements for intraretinal layers measured using the Spectralis OCT system. There were no significant differences among the three thickness measurements obtained by the same examiner. ICCs obtained for the intraobserver repeatability and interobserver reproducibility tests were >0.964 for the entire retina and for all of the intraretinal layers in all of the subfields (Table 1).

Table 1 Intraobserver and interobserver agreement for retinal layer thickness 

T-a1 (µm) T-a2 (µm) T-a3 (µm) T-b (µm) ICCa1-2 ICCa1-3 ICCa-b
RNFL 011.62 ± 02.48 011.59 ± 02.51 011.69 ± 02.55 011.24 ± 02.38 1.000 1.000 0.971
GCL 013.74 ± 03.93 013.55 ± 03.71 013.92 ± 03.98 013.43 ± 03.86 0.982 0.989 0.975
IPL 019.94 ± 03.31 019.90 ± 03.27 019.86 ± 03.25 019.82 ± 03.11 1.000 0.997 1.000
INL 015.84 ± 04.52 015.69 ± 04.41 015.88 ± 04.49 015.83 ± 04.34 0.989 1.000 1.000
OPL 021.58 ± 04.50 021.65 ± 04.61 021.55 ± 04.57 021.54 ± 04.39 0.991 1.000 1.000
ONL 091.97 ± 09.51 091.84 ± 09.52 091.68 ± 09.42 092.36 ± 09.51 1.000 0.974 1.000
RPE 016.22 ± 02.19 016.22 ± 02.13 016.10 ± 02.19 016.22 ± 02.19 1.000 0.985 1.000
IRL 181.07 ± 22.70 182.36 ± 23.26 182.79 ± 22.88 181.85 ± 22.21 1.000 1.000 1.000
ORL 085.91 ± 06.04 084.64 ± 05.18 084.82 ± 06.04 084.34 ± 05.71 1.000 1.000 1.000
İnner ring
RNFL 011.62 ± 02.48 011.59 ± 02.51 011.69 ± 02.55 011.24 ± 02.38 1.000 1.000 0.971
GCL 013.74 ± 03.93 013.55 ± 03.71 013.92 ± 03.98 013.43 ± 03.86 0.982 0.989 0.975
IPL 019.94 ± 03.31 019.90 ±0 3.27 019.86 ± 03.25 019.82 ± 03.11 1.000 0.997 1.000
INL 015.84 ± 04.52 015.69 ± 04.41 015.88 ± 04.49 015.83 ± 04.34 0.989 1.000 1.000
OPL 021.58 ± 04.50 021.65 ± 04.61 021.55 ± 04.57 021.54 ± 04.39 0.991 1.000 1.000
ONL 091.97 ± 09.51 091.84 ± 09.52 091.68 ± 09.42 092.36 ± 09.51 1.000 0.974 1.000
RPE 016.22 ± 02.19 016.22 ± 02.13 016.10 ± 02.19 016.22 ± 02.19 1.000 0.985 1.000
IRL 181.07 ± 22.70 182.36 ± 23.26 182.79 ± 22.88 181.85 ± 22.21 1.000 1.000 1.000
ORL 085.91 ± 06.04 084.64 ± 05.18 084.82 ± 06.04 084.34 ± 05.71 1.000 1.000 1.000
Outer ring
RNFL 035.87 ± 03.24 036.08 ± 03.81 035.56 ± 03.20 036.21 ± 03.42 0.989 0.987 0.971
GCL 037.83 ± 03.25 037.30 ± 03.08 038.11 ± 03.46 037.85 ± 03.30 0.975 0.980 1.000
IPL 030.41 ± 02.45 030.35 ± 02.34 030.45 ± 02.45 030.40 ± 02.49 1.000 1.000 1.000
INL 033.61 ± 02.01 033.58 ± 02.00 033.44 ± 01.97 035.04 ± 02.32 1.000 0.998 0.964
OPL 027.72 ± 02.17 027.69 ± 02.10 027.57 ± 02.05 027.70 ± 02.17 1.000 0.996 1.000
ONL 058.89 ± 06.51 058.98 ± 06.66 058.75 ± 06.49 058.83 ± 06.51 1.000 1.000 1.000
RPE 012.69 ± 01.23 013.03 ± 01.25 012.88 ± 01.23 013.05 ± 01.47 0.979 0.996 0.979
IRL 222.18 ± 10.51 222.78 ± 10.44 222.10 ± 10.50 223.20 ± 09.42 1.000 1.000 1.000
ORL 077.17 ± 01.97 077.42 ± 02.79 078.47 ± 01.86 076.75 ± 01.78 1.000 1.000 1.000

T-a1= mean thickness of first measurement by examiner 1; T-a2= mean thickness of second measurement by examiner 1; T-a3= mean thickness of third measurement by examiner 1; T-b= mean thickness of first measurement by examiner 2; ICC a1-2= intraclass correlation coefficients of repeatability (T-a1-T-a2); ICC a1-3= intraclass correlation coefficients of repea­tability (T-a1-T-a3); ICCa-b= intraclass correlation coefficients of reproducibility; RNFL= retinal nerve fiber layer; GCL= ganglion cell layer IPL; inner plexiform layer; INL= inner nuclear layer; OPL= outer plexiform layer; ONL= outer nuclear layer; RPE= retinal pigment epithelium; IRL= inner retinal layer; ORL= outer retinal layer.


Repeatability and reproducibility are very important for assessing measurement fluctuations for an instrument and when an instrument is used by multiple operators. Measurements of the intraretinal layers are significant morphometric parameters in the diagnosis of retinal and neurological diseases as well as for monitoring disease progression(13). In this study, we evaluated the repeatability and reproducibility of measurements for nine intraretinal layers (RNFL, GCL, IPL, INL, OPL, ONL, RPE, IRL, ORL) determined by an automated algorithm applied to images obtained using the SD-OCT system with the follow-up mode. The follow-up mode is also called AutoRescan and implements active eye tracking to perform OCT scans automatically on the retina at the same point as in the previous review(14). In our study, for both intraobserver and interobserver comparisons, ICCs were high for all layers. The intraobserver and interobserver test results indicated that the SD-OCT system produced excellent repeatable and reproducible measurements for all of the intraretinal layers. The repeatability of measurements in the present study is consistent with those in previous reports involving different OCT devices.

Previous studies reported a higher degree of repeatability for macular thickness with Spectralis OCT measurements. Fiore et al. demonstrated that the eye tracking mechanism and follow-up function improved reproducibility, ensuring the same scanning location was selected on following visits and that moving artifacts were reduced(15). Ctori et al. reported excellent repeatability and reproducibility for the thickness of each of eight individual retinal layers located centrally or at 2° or 5° eccentricity away from the foveal measurements obtained using the SD-OCT segmentation software in a young healthy cohort(16). What differentiates our study from previous studies is its assessment of repeatability and reproducibility in all subdivisions (wide area) of ETDRS. Debuc et al. reported the repeatability and reproducibility of thickness measurements for six intraretinal layers using automated custom software developed for the Stratus OCT with healthy subjects. They found that ICCs were >0.75 for all layers except for the OPL and OS/RPE(17). Wang et al. reported the repeatability of thickness measurements for nine intraretinal layers determined by manual segmentation of ultra-high-resolution OCT (UHR-OCT) images with an axial resolution of approximately 2 µm with healthy subjects. The ICC was >0.90 in most of the intraretinal layers(18). Liu et al. reported the repeatability and reproducibility of thickness measurements for eight-retinal layers using a custom-built UHR-OCT instrument (~3 µm resolution) and a commercial RTVue100 OCT (~5 µm resolution) instrument with 20 normal subjects. They found ICCs of >0.80 for all layers except for the inner and outer segments(13). Our results are consistent with those of these studies, showing that the thickness measurements of higher-axial-resolution OCT instruments have better repeatability for all retinal layers.

There are some limitations to the present study. First, it included only healthy subjects. Diseased retinal structures may vary substantially among patients, which is likely to increase the frequency of segmentation errors. Thus, the repeatability and reproducibility values may be reduced in diseased retinas. In future studies, we will apply our new method to evaluate a variety of retinal diseases. However, the purpose of the current study was to determine the clinical significance of any changes in the repeatability and reproducibility of the intraretinal measurements.

In conclusion, the current study evaluated measurements of retinal layer thickness in the macula area. The measurements obtained using the follow-up mode of the SD-OCT system demonstrated high repeatability and reproducibility with excellent ICCs.

Funding: No specific financial support was available for this study.

Approved by the following research ethics committee: Afyon Kocatepe University (# 2017/4).


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Received: April 25, 2017; Accepted: July 17, 2017

Corresponding author: Ersan Çetinkaya. Department of Ophthalmology. Manisa State Hospital. Manisa, Turkey -

Disclosure of potential conflicts of interest: None of the authors have any potential conflict of interest to disclose.

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