Repeatability of measuring the vessel density in patients with retinal vein occlusion: An optical coherence tomography angiography study.

PURPOSE: To determine the repeatability of superficial vessel density measurements using Spectral domain Ocular coherence tomography angiography(SD-OCTA) in patients diagnosed with retinal vein occlusion(RVO).
DESIGN: Prospective observational study.
SUBJECTS: Patients who visited our retinal clinic from August 2017 to August 2018, diagnosed with RVO were recruited for the study.
METHODS: Two consecutive 3×3 mm pattern scans were performed using the Cirrus HD-OCT 5000 along with AngioPlex software (Carl Zeiss Meditec) in each eye by single skilled examiner. All scans were analyzed using en face OCTA images to measure vessel density (VD) automatically. For further analysis of the effect of central macular thickness(CMT), eyes were divided into two groups according to CMT of 400μm (Group 1: CMT > 400μm, Group 2: CMT < 400μm). To identify factors affecting the repeatability of VD measurements, linear regression analyses were conducted for the coefficient of variation (CV) of VD by investigating demographics and ocular variables. MAIN OUTCOME MEASURES: The intraclass correlation coefficient (ICC), coefficient of variation (CV) of VD measurements.
RESULTS: A total of 57 eyes from 57 patients were examined: 35 eyes with BRVO and 22 eyes with CRVO. In all 57 eyes with RVO, the ICC and CV of the full VD(VD of 3mm diameter circle) were 0.800 and 10.61%, respectively. Univariate analyses showed that the mean CMT (B, 0.001; p<0.001) and mean ganglion cell-Inner plexiform layer (GC-IPL) thickness (B, -0.002; p = 0.020) were significant factors that affected the repeatability. Multivariate analyses of these two factors showed that only mean CMT was a significant factor. The ICC and CV of the full VD in group 1 (CMT > 400μm) were 0.348 and 22.55% respectively. In group 2 (CMT < 400μm), the ICC and CV of the full VD were 0.910 and 7.76%, respectively.
CONCLUSIONS: The repeatability of VD measurement in eyes with RVO was reasonably comparable to previous studies. Repeatability of VD measurement was significantly affected by central macular thickness.

Introduction

Retinal vein occlusion (RVO) is the second most common retinal vascular disease after diabetic retinopathy. Nearly 2% of people over 40 years of age are diagnosed with RVO [1, 2]. RVO can cause various degrees of vision problems in the older worldwide population. Given the prevalence of the aging population throughout the world, the accurate diagnosis and adequate management of RVO have become critically important [3-6].

Fluorescein angiography (FA) has been the method primarily chosen for assessing vascular abnormalities associated with RVO. However, FA has many limitations because it is a time-consuming and invasive procedure, and the diffusion of dye makes it difficult to observe the microvasculature in the late phase of the exam. In contrast, optical coherence tomography angiography (OCTA) is noninvasive and less time-consuming, and it provides depth-resolved images to visualize the retinal vasculature in multiple layers. OCTA also provides quantitative metrics of the retinal microvasculature such as vessel density (VD), perfusion density, and the foveal avascular zone (FAZ) area of the retinal capillary plexus [7-11]. Even though OCTA has several limitations such as projection artifacts and narrow field of view, this novel technique can provide clinicians with microvascular information that can assist diagnosis and treatment of many types of retinal vascular diseases [12, 13].

Many studies reported microvascular changes in eyes with RVO using OCTA [3,14,15]. At the same time, the reliability and efficacy of the OCTA have been questioned. The repeatability of this new device have been reported in many studies in normal eyes. However, there is limited study on the repeatability of OCTA in eyes with RVO. In the present study, we determined the repeatability of VD measurements using the Cirrus HD-OCT 5000 with AngioPlex software (version 10.0; Carl Zeiss Meditec, Dublin, CA, USA) in patients diagnosed with RVO. We also identified factors affecting the repeatability of VD measurements.

Materials and methods

Patients

This study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Chungnam National University Hospital (Daejeon, Republic of Korea). The study was performed prospectively using patients who were diagnosed with RVO who visited our retinal clinic from August 2017 to August 2018. Patients were diagnosed with RVO through comprehensive examinations including funduscopy, OCT and FA. Patients were excluded if they had any intraocular surgery except for cataract surgery. Each patient gave a detailed history and underwent testing for best-corrected visual acuity (BCVA), intraocular pressure using noncontact tonometry, spherical equivalent, axial length using an IOL Master (Carl Zeiss, Jena, Germany), and the mean central macular thickness (CMT) and ganglion cell-inner plexiform layer (GC-IPL) thickness using a Cirrus HD-OCT 5000 (Carl Zeiss Meditec).

OCTA measurements

Two consecutive measurements were performed by a single skilled examiner using the Cirrus HD-OCT 5000 along with AngioPlex software (Carl Zeiss Meditec). The AngioPlex used a center wavelength of 840 nm, taking 68,000 A-scans/s to obtain high-resolution microvascular images. The instrument is based on the optical microangiography (OMAG) algorithm, and the retinal tracking program helps provide high sensitivity and accuracy. We used a 3 × 3 mm pattern scan to measure the central foveal area, and all scans were analyzed using en face OCTA images generated automatically by the OMAG algorithm used in the AngioPlex software. The VD (defined as the total length of perfused vasculature per unit area in the region of measurement) of the superficial layer was measured automatically by the software, which quantitated the VD of a local region of tissue according to the Early Treatment of Diabetic Retinopathy Study(ETDRS) subfields. The superficial layer was defined as the layer starting from the internal limiting membrane (ILM) to the inner plexiform layer (IPL). The IPL boundary was calculated as 70% of the distance from the ILM to the estimated boundary of the outer plexiform layer, which was determined to be 110 μm above the retinal pigment epithelium boundary as automatically detected by the software. AngioPlex software also measures the foveal avascular zone(FAZ) metrics automatically. It provides the measurement of FAZ area and FAZ perimeter. To analyze the repeatability of VD measurements using the automatic AngioPlex software, we did not make any manual adjustments. In this study, we analyzed the VD of the full area (3 mm diameter ring), inner area (1mm diameter inner ring), and each sector of ETDRS subfields. We also analyzed the FAZ area and FAZ perimeter. We excluded OCTA images with a signal strength < 7.

Statistical analyses

To analyze the repeatability of the VD in patients with RVO, we calculated the intraclass correlation coefficient (ICC), coefficient of variation (CV), and test-retest standard deviation (TRTSD) of the full VD. An ICC (the ratio of the subject variance to the total variance) close to 1 meant that the variance was low in the same examination. The CV (%) was calculated as 100 × SD/overall mean, and a value < 10% meant good repeatability of the measurement. TRTSD was calculated as the square root of the within-subject mean square for error. The agreement between two measurements was evaluated using Bland-Altman plots. To identify factors affecting the repeatability of VD measurements, linear regression analyses were con-ducted for the CV of the full VD by investigating demographic and ocular variables. Multi-variate analyses were performed for significant values of p < 0.05 obtained by univariate analyses.

Results

Demographics and main criteria

A total of 62 eyes from 62 patients were recruited. 5 patients were excluded for poor image quality (signal strength < 7). Finally, 57 eyes from 57 patients were examined: 35 eyes with BRVO and 22 eyes with CRVO. The mean age was 65.6 years, the mean BCVA was 0.34 the mean spherical equivalent was +0.14, the mean axial length was 25.5 mm, the mean CMT was 353.7 μm, and the mean GC-IPL thick-ness was 53.7 μm (Table 1). Baseline characteristics of subgroups divided according to CMT of 400 μm is shown in Table 2. There were no significant differences between the two groups except for mean CMT.

Table 1

Demographics and baseline characteristics of patients.

Number of subjects	57
Branch retinal vein occlusion	35
Central retinal vein occlusion	22
Age (years, mean±SD)	65.6±9.1
Male gender (%)	42.1
Diabetes mellitus (%)	24.6
Hypertension (%)	42.1
Right laterality (%)	45.6
Phakic eye (%)	80.7%
BCVA (logMAR, mean±SD)	0.34±0.34
SE (dioptres, mean±SD)	+0.14±1.69
IOP (mmHg, mean±SD)	16.3±2.8
Axial length (mm, mean±SD)	23.5±0.8
Mean signal strength (mean±SD)	8.6±1.2
Mean CMT (μm, mean±SD)	353.7±139.4
Mean GC-IPL thickness (μm, mean±SD)	53.7±29.2

BCVA, best corrected visual acuity; SE, spherical equivalent; IOP, intraocular pressure; CMT, central macular thickness; GC-IPL, ganglion cell-inner plexiform layer.

Table 2

Demographics and baseline characteristics of each group.

	Group 1 (CMT>400)	Group 2 (CMT<400)
Number of subjects	16	41
Age (years, mean±SD)	64.8±9.7	66.0±8.9
BCVA (logMAR, mean±SD)	0.39±0.28	0.31±0.36
SE (dioptres, mean±SD)	+0.31±2.19	+0.07±1.49
IOP (mmHg, mean±SD)	16.6±2.9	16.2±2.8
Axial length (mm, mean±SD)	23.4±0.9	23.6±0.8
Mean signal strength (mean±SD)	8.1±1.2	8.8±1.1
Mean CMT (μm, mean±SD)	544.7±105.4	279.1±52.5
Mean GC-IPL thickness (μm, mean±SD)	20.1±12.9	66.9±22.3

Group 1: Eyes with central macular thickness greater than 400 μm; Group 2: Eyes with central macular thickness lesser than 400 μm.

BCVA, best corrected visual acuity; SE, spherical equivalent; IOP, intraocular pressure; CMT, central macular thickness; GC-IPL, ganglion cell-inner plexiform layer.

Repeatability of the VD in RVO patients

In all 57 eyes with RVO, the ICC and CV of the full VD were 0.800 and 10.61%, respectively. In each sector, the ICC of the center, superior, nasal, inferior, and temporal areas were 0.755, 0.843, 0.824, 0.855, and 0.758, respectively, and the CV of the center, superior, nasal, inferior, and temporal areas were 21.48%, 10.30%, 10.59%, 11.88%, and 11.17%, respectively (Table 3). Using a Bland-Altman plot, the difference was close to zero in the measurement of VD (Fig 1).

Table 3

First and second mean values, intraclass correlation coefficient, coefficient of variation and test-retest standard deviation of vessel density in patients with RVO.

	First mean VD	Second mean VD	ICC	CV (%)	TRTSD
Full	15.74±4.2	16.02±3.9	0.800	10.61	1.06
Inner	16.83±4.3	17.21±3.9	0.852	8.28	0.89
Sectorial
Central	8.3±3.6	8.7±3.7	0.755	21.48	1.09
Superior	17.0±5.0	16.9±4.4	0.843	10.30	1.08
Nasal	17.7±4.8	17.9±4.7	0.824	10.59	1.06
Inferior	15.9±5.4	16.5±5.0	0.855	11.88	1.15
Temporal	16.6±4.7	17.7±4.1	0.758	11.17	1.20

CV, coefficient of variation; ICC, intraclass correlation coefficient; TRTSD, test-retest standard deviation; VD, vessel density.

Fig 1

Bland-Altman plots showing the level of agreement for the VD measurements obtained using AngioPlex optical coherence tomography between two consecutive measurements in patients with RVO.

VD measurements of 7 different areas: (1)Full area, (2) Inner area, (3)Central, (4)Superior, (5)Nasal, (6)Inferior, (7)Temporal. Two dot lines indicate the upper and lower boundaries of the 95% CIs. VD, vessel density.

In FAZ analysis, we had to exclude 20 eyes which AngioPlex software could not detect the FAZ. Then we reviewed the FAZ line that was drawn automatically by the Angioplex software in 37 eyes. Among 37 eyes, the FAZ line was inappropriate in 13 eyes. FAZ line was drawn either too small or away from the fovea. Therefore we couldn’t investigate the repeatability of the FAZ metrics in this study.

Factors affecting the repeatability of VD measurements

Univariate analyses showed that the mean CMT (B: 0.001; p < 0.001) and mean GC-IPL thickness (B: -0.002; p = 0.020) were significant factors that affected the repeatability. Multivariate analyses of these two factors showed that only the mean CMT was a significant factor (Table 4). Fig 2 shows a scatterplot graph of the VD differences in the average CMT between two consecutive measurements. Eyes with large central macular thicknesses showed a relatively large difference compared to eyes with normal central macular thicknesses, which showed a small difference.

Table 4

Univariate and multivariate linear regression for the association between clinical and anatomical parameters and coefficient of variation of vessel density.

	Univariate		Multivariate
	B (95% CI)	P values	B (95% CI)	P values
Age	-0.002(0.003)	0.577
Sex	-0.012(0.043)	0.778
Laterality	-0.001(0.042)	0.989
Phakic eye	0.010(0.061)	0.865
Diabetes	-0.009(0.048)	0.849
Hypertension	-0.061(0.048)	0.204
SE	-0.010(0.015)	0.487
BCVA	0.053(0.081)	0.519
IOP	-0.002(0.008)	0.833
Axial length	-0.034(0.033)	0.315
Mean signal strength	0.016(0.025)	0.518
Mean CMT	0.001(0.000)	0.003	0.001(0.000)	0.002
Mean GC-IPL thickness	-0.002(0.001)	0.020	0.000(0.001)	0.877

Values with p<0.05 are shown in bold.

BCVA, best corrected visual acuity; CMT, central macular thickness; GC-IPL, ganglion cell-inner plexiform layer; IOP, intraocular pressure; SE, spherical equivalent.

Fig 2

Scatterplot graph of the differences in VD between two consecutive measurements using optical coherence tomography angiography.

The differences in VD tended to be larger when the CMT was thicker. CMT, central macular thickness; VD, vessel density.

We also conducted a subgroup analysis by dividing the eyes into two groups according to a CMT of 400 μm. We separately analyzed the ICC, CV, and TRTSD in the two groups. As a result, the ICC and CV of the full VD in group 1 (CMT > 400 μm) were 0.348 and 22.55%, respectively. In group 2 (CMT < 400 μm), the ICC and CV of the full VD were 0.910 and 7.76%, respectively (Table 5).

Table 5

Subgroup analysis by CMT value of 400 μm.

First and second mean values, intraclass correlation coefficient, coefficient of variation and test-retest standard deviation of vessel density in patients with RVO.

		First mean VD	Second mean VD	ICC	CV (%)	TRTSD
CMT>400 μm	Full	14.9±4.5	16.5±3.0	0.348	22.55	1.83
	Inner	16.0±4.5	17.3±3.2	0.439	13.47	1.35
	Sectorial
	Central	9.36±3.9	11.2±3.1	0.235	29.24	1.82
	Superior	15.1±5.8	16.6±4.4	0.668	17.18	1.58
	Nasal	17.1±4.7	18.7±2.8	0.519	12.07	1.29
	Inferior	15.7±5.7	16.3±5.6	0.757	15.47	1.43
	Temporal	15.4±5.5	18.0±3.1	0.353	20.76	2.00
CMT<400 μm	Full	16.1±4.1	15.9±4.2	0.910	7.76	0.80
	Inner	17.2±4.3	17.2±4.2	0.946	6.26	0.71
	Sectorial
	Central	7.83±3.5	7.79±3.6	0.863	18.46	0.81
	Superior	17.8±4.6	17.1±4.4	0.925	7.61	0.89
	Nasal	17.9±4.9	17.6±5.3	0.886	10.02	0.97
	Inferior	16.0±5.3	16.6±4.9	0.895	10.48	1.04
	Temporal	17.1±4.3	17.6±4.5	0.881	7.42	0.89

CV, coefficient of variation; ICC, intraclass correlation coefficient; TRTSD, test-retest standard deviation; VD, vessel density.

Discussion

Since its introduction, many studies have used OCTA to analyze eyes with RVO. Rispoli et al [14] reported that OCTA detected foveal avascular zone enlargement, capillary nonperfusion, microvascular abnormalities, and vascular congestion signs both in the superficial and deep capillary network in all eyes affected by RVO. Suzuki et al. [15] also reported that the visualization of microvascular abnormalities in eyes with macular edema associated with RVO was equal or better using OCTA compared to FA. Lee et al. [16] showed that the repeatability of VD measurements using OCTA in various retinal diseases, including RVO, was relatively good. However, to the best of our knowledge, no studies have been reported that analyzed the repeatability of OCTA measurements extensively and exclusively in eyes with RVO.

Previous studies have reported good repeatability and reproducibility of OCTA in normal eyes [7-10]. Lee et al. [16] reported that the CV of the full VD was 10.65% in eyes with RVO, which was higher than those with other retinal diseases. In our study, the CV of the full VD was 10.61%, which was comparable. In both studies, the CV of the central area was higher than that of other areas. The reason is probably because the CV was affected by the average. The CV was calculated as 100 × SD/overall mean, so the central area had a relatively lower VD than other areas. Additionally, Parodi et al. [17] reported an enlarged FAZ area in eyes diagnosed with RVO using FA. Other studies also reported an enlarged FAZ area using OCTA, consistent with previous findings [14, 15]. Therefore, a decreased VD measurement in the central area could be explained by an enlarged FAZ area (Table 3). This is perhaps the reason why the CV of the central area was higher than that of other areas. When analyzing the CV in eyes with RVO, we therefore need to consider the effect of an enlarged FAZ area, which can affect the VD average and the CV.

According to a univariate linear regression analysis, the mean CMT and GCIPL thickness were two factors affecting the repeatability of VD measurements. Lee et al. [16] suggested that in univariate linear regression analyses of the BCVA, the mean signal strength, mean CMT, and mean GCIPL thicknesses were significant factors affecting the repeatability of the VD measurement in various retinal diseases, including RVO, but a multivariate analysis showed that the GC-IPL thickness was the only factor. In the present study, GC-IPL thickness was also a significant factor using the univariate model. GC-IPL thickness was negatively correlated with the CV of VD measurements. The VD could have been decreased in eyes with a thinner GC-IPL, which may have caused the CV of VD measurements to increase. However, using multivariate analyses, the GC-IPL was not significant (p = 0.877). In eyes with macular edema, OCTA tends to measure a thinner GCIPL thickness than the actual thickness because of segmentation error [18, 19]. Therefore, the impact of the actual GCIPL thickness on the repeatability of OCTA might have been diminished; additional studies are needed to prove this hypothesis.

The only significant factor in the multivariate regression analyses was the mean CMT, which was negatively related with the repeatability of VD measurements. The mean CMT can affect VD measurements in three ways. First, in eyes with macular edema, the normal contour of the retinal layers is distorted and segmentation errors could easily occur, so it can significantly affect the VD measurements. In our study we discovered 7 eyes with definite segmentation error and all 7 eyes were affected by macular edema. Second, patients with macular edema had lower visual acuity. There might have been difficulties in achieving proper fixation when examining the eye. Lastly, macular edema can overshadow the retinal vasculature, which may also alter VD measurements and affect their repeatability. Interestingly, recent study by Nicolai et al suggest the application of OCTA on peripapillary area to evaluate microvascular changes when the CRVO is complicated with macular edema. According to their study, peripapillary metrics can be more reliable data when the macular metrics are unreliable due to macular edema [20].

To further examine the effect of CMT in VD measurements, we conducted a subgroup analysis dividing the eyes into two groups according to a CMT of 400 μm. Subgroup analysis showed a distinct difference between the two groups in the repeatability of VD measurements. In group 1 (CMT > 400 μm), the ICC and CV were 0.348 and 22.55%, respectively, compared to 0.910 and 7.76%, respectively, in group 2. The repeatability was significantly lower in eyes with severe macular edema. Previously, many OCTA studies of RVO patients have included analyses of eyes with macular edema. These studies did not consider the effect of macular edema on VD measurements, which needs to be reconsidered because the OCTA measurements may have not been reliable [21, 22]. Also, clinicians should be aware of the effect of macular edema when interpreting OCTA data, especially when the CMT is > 400 μm.

BRVO can occur in different regions according to the site of the occlusion. We expected lower repeatability of VD measurements in the area involved with BRVO region and analyzed 35 eyes with BRVO. However, the CV of VD measurement in involved and uninvoled areas were 9.2% and 10.5% respectively, which did not show definite difference. The results can be explained by the effect of macular edema. In our study 26 eyes (74%) had macular edema and in most of the eyes with macular edema, all of the ETDRS inner circle area, which we analyzed in this study, were affected by the edema regardless of the BRVO region. Further studies analyzing OCTA scans with wider area are needed.

Foveal avascular zone enlargement in eyes with RVO is a well-known phenomenon. Parodi et al. [17] reported enlargement of FAZ in BRVO using FA and its high correlation with visual impairment. Other studies also reported similar FAZ changes using OCTA [14,15]. Therefore the reliability of OCTA in detecting FAZ is a important factor to consider when evaluating patients with RVO. In our study we couldn’t analyze the repeatability of FAZ metrics. Angioplex software failed to detect FAZ in 20 eyes. Additionally, 13 eyes had inappropriate FAZ line that was drawn automatically by the software. The automated FAZ measurements weren’t reliable and manual measurements would be neccessary when analyzing FAZ metrics in eyes with RVO.

There are several limitations to this study. We only used 3 × 3 mm scan images for analysis. Currently, more pattern scan settings are provided by the OCTA. However, according to a previous study, 3 × 3 mm scans obtained better image resolution than 6 × 6 mm scans when measuring the superficial retinal VD, making it more appropriate for analysis [23]. We could not analyze the VD of the deep retinal layer because the AngioPlex software detected only the VD of the superficial retinal layer. However, due to projection artifacts it is known that an analysis of the superficial retinal layer is more accurate than that of the deep retinal layer. We only scanned the OCTA twice per each eye to investigate repeatability due to the limitation of clinical situation. Finally, we could not analyze the data after manual segmentation because current version of AngioPlex software does not provide quantitative data when manual segmentation is performed. We need further study on the reliability of manual segmentation in OCTA.

In conclusion, OCTA is a practical tool for evaluating eyes with RVO, and it can be used for various clinical measurements. However, when analyzing the OCTA results in eyes with RVO associated with macular edema, clinicians should consider the possibility of its lower repeatability and reliability.

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2 Apr 2020

PONE-D-20-06442

Repeatability of measuring the vessel density in patients with retinal vein occlusion: an optical coherence tomography angiography study.

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Reviewer #1: Comments for the Authors: PONE-D-20-06442

1) General comments

The authors assessed the repeatability of the vessel density measurements by the OCTA, however, the authors did not clarify what have been investigated so far and what kind of unsolved research questions are going to be tested in the study. Also, the flow of the sentences is usually off and hard to understand the sales point of the manuscript.

Specific comments:

- Abstract: Please spell out several abbreviations (e.g. SD-OCTA, VD, CV etc.) at their first appearance in the abstract.

- Abstract: lease specify what the full VD means.

- Abstract: The terms "group 1" and "group 2" were suddenly come up in the Results. Please define those in the Methods properly.

- Introduction: The authors should reconsider the flow of sentences in the introduction, which do not sound well and hard to understand. For example, the introduction said that RVO were classified into two categories.., but this information is not related to the following sentence and would be redundant.

- Introduction: The authors stated OCTA has provided many benefits though there are many limitations in FA - however, the authors did not explain what are the "many benefits" of OCTA and not only pros but also their cons should be explained.

- Introduction: What is the sale point of the study? - Could not find any new information/investigations from the study. What is the strong point of the study, which has not been investigated?

- Introduction: The authors did not put appropriate citations throughout the introduction section. Please cite previous paper which support what the authors stated.

- Methods: Please provide the diagnosing criteria for RVO - did the atuhors use FA or just an OCTA?

- Methods: Methods: Did the authors choose one eye per patient or sometimes both eyes? If both eyes were used, specific stat models such as GLM/ GEE should be considered to employ.

- Methods: Demographics should be also shown by CMT category, i.e., <400 and >400 groups.

Reviewer #2: The manuscript by Dr. Kim et al investigated the repeatability of vessel density measurement in RVO patients and explore its related factor. It found that in eyes with macular edema, the repeatability is poor. Generally it is interesting, but further revisions are needed.

1. Please clarify how the sample size be calculated. This study only repeated the scan twice. Why didn't you scan more times?

2.This study found that macular edema is a risk factor of poor repeatability, it also give some explaination in the discussion. I would like to suggest the author to quantifiy segmentation error in their images and report the repeatability after manual correction of segmentation.

3. This study only reported the results of vessel density. The metrics of foveal avascular zone are also important paramters on OCTA. Please also investigate the repeatability of FAZ metrics.

4. The study subjects are mixed of CRVO and BRVO. There are also different region of BRVO. It would be interesting to investigate whether the repeatability is different between the region involved and not involved.

Reviewer #3: The authors present an assessment of OCTA repeatability in retinal vein occlusion.

The authors also report a linear regression analysis of clinical and anatomical parameters affecting VD repeatibility and demonstrate how the increase of macular volume reduce the accuracy of the examination.

Our group recently published an angio-OCT study to evaluate Papillary Vessel Density Changes after Intravitreal Anti-VEGF Injections in Central Retinal Vein Occlusion (J Clin Med. 2019 Oct 6;8(10). pii: E1636. doi: 10.3390/jcm8101636). We believe that the peripapillary area might be a region of interest to study microvascular changes when significant macular edema is present. I think it would be interesting to add a comment on this subject.

In the conclusions section it would be more appropriate to specify that there is a correlation between the entity of macular edema and the repeatability of VD measurement rather than stating that the repeatability is "poor when eyes were associated with macular edema".

Minor corrections:

Page 13 line line 120: typos thickness

Page 15 line 220 lower instead of low

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Reviewer #2: Yes: Haoyu Chen

Reviewer #3: Yes: Michele Nicolai

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17 May 2020

Reviewer #1: Comments for the Authors: PONE-D-20-06442

1) General comments

The authors assessed the repeatability of the vessel density measurements by the OCTA, however, the authors did not clarify what have been investigated so far and what kind of unsolved research questions are going to be tested in the study. Also, the flow of the sentences is usually off and hard to understand the sales point of the manuscript.

: Thank you for your sincere review. We agree the flow of the manuscript seems unnatural and the sale point was mentioned vaguely. We have refined the sentences and emphasized the strong point of the study in the introduction.

Specific comments:

- Abstract: Please spell out several abbreviations (e.g. SD-OCTA, VD, CV etc.) at their first appearance in the abstract.

- Abstract: Please specify what the full VD means.

- Abstract: The terms "group 1" and "group 2" were suddenly come up in the Results. Please define those in the Methods properly.

: Thank you for your comments. We have spelled out the abbreviations at their first appearance. We have explained the meaning of full VD in its first appearance in the abstract. We have added the definition of the subgroups in the method section in the abstract.

- Introduction: The authors should reconsider the flow of sentences in the introduction, which do not sound well and hard to understand. For example, the introduction said that RVO were classified into two categories.., but this information is not related to the following sentence and would be redundant.

: Thank you for your comment. We have read the Introduction thoroughly considering the flow of sentences. We have erased the sentence stating the classification of RVO that is thought to be redundant. Considering the flow we revised the sentences in the introductions. (Page 4, line 61-64, 65-68)

- Introduction: The authors stated OCTA has provided many benefits though there are many limitations in FA - however, the authors did not explain what are the "many benefits" of OCTA and not only pros but also their cons should be explained.

: Thank you for your comment. We have stated the benefits of OCTA in the introduction. “OCTA is noninvasive and less time-consuming, and it provides depth-resolved images to visualize the retinal vasculature in multiple layers. OCTA also provides quantitative metrics of the retinal microvasculature”. (Page 4, line 57-61) Even though we have stated the pros of the OCTA, for better understanding we have added the cons of OCTA in the following sentence. “Even though OCTA has several limitations such as projection artifacts and narrow field of view, this novel technique can provide clinicians with microvascular information that can assist diagnosis and treatment of many types of retinal vascular diseases.” (Page 4, line 61-64)

- Introduction: What is the sale point of the study? - Could not find any new information/investigations from the study. What is the strong point of the study, which has not been investigated?

: We agree with your comment. We have stated the sale point of this study in the introductions but it seems ambiguous. For concise expression we have revised and added following sentences. ‘Many studies reported microvascular changes in eyes with RVO using OCTA. (3,14,15) At the same time, the reliability and efficacy of the OCTA have been questioned. The repeatability of this new device have been reported in many studies in normal eyes, however, there is limited study on the repeatability of OCTA in eyes with RVO.’ (Page 4, line 65-68)

- Introduction: The authors did not put appropriate citations throughout the introduction section. Please cite previous paper which support what the authors stated.

: Thank you for your comment. We have added total of 15 references to support the statements made in the introduction section.

- Methods: Please provide the diagnosing criteria for RVO - did the authors use FA or just an OCTA?

: Thank you for your suggestion. We diagnosed RVO through funduscopy exam, OCT and FA findings. We have added the following statement in the method section (page 5, line 78-79)

- Methods: Methods: Did the authors choose one eye per patient or sometimes both eyes? If both eyes were used, specific stat models such as GLM/ GEE should be considered to employ.

: Thank you for your comment. We chose one eye per patient in this study. To avoid the confusion we have stated in the Demographics ‘A total of 57 eyes from 57 patients were examined’.(page 7, line 120-121)

- Methods: Demographics should be also shown by CMT category, i.e., <400 and >400 groups.

: Thank you for your comment. We have added table 2 for demographics of each subgroups. There were no significant characteristic differences between the two group except for CMT. We added the following statement in the demographics. (Page 7, line 124-126)

Reviewer #2: The manuscript by Dr. Kim et al investigated the repeatability of vessel density measurement in RVO patients and explore its related factor. It found that in eyes with macular edema, the repeatability is poor. Generally it is interesting, but further revisions are needed.

1. Please clarify how the sample size be calculated. This study only repeated the scan twice. Why didn't you scan more times?

: Thank you for your comment. Thank you for your comment. We did not perform sample size calculations in this study. We tried to include more than 50 eyes so that various variables including within subject standard deviation can follow the normal distribution. We have reviewed every clinical record from August 2017 to August 2018 retrospectively. Total of 62 patients were recruited for the study and 5 patients were excluded due to poor image quality.

Most of the patients in this study were old aged and scanning multiple times can cause fatigue and dryness of the eyes. Therefore, we were limited to scan patients twice since it was a retrospective study based on clinical situation. We have added the following limitation in the discussion.

Most of the patients in this study were old aged and scanning multiple times can cause fatigue and dryness of the eyes. Therefore, we were limited to scan patients twice since it was a retrospective study based on clinical situation. We have added the following limitation in the discussion.

2. This study found that macular edema is a risk factor of poor repeatability, it also give some explanation in the discussion. I would like to suggest the author to quantify segmentation error in their images and report the repeatability after manual correction of segmentation.

: Thank you for your suggestion. This study is aimed to investigate the factors that can affect the OCTA result by autosegmentation which we mostly encounter in the clinic.We have identified 7 eyes with definite segmentation errors and all eyes were affected by macular edema. We highly agree with your comment on the need for manual segmentation. However we couldn’t perform the manual segmentation because the current version of OCTA software cannot measure the VD when manual segmentation is performed afterwards. We have added the following information in the discussion and limitation. (Page 15, line 225-226, page 17 266-268)

3. This study only reported the results of vessel density. The metrics of foveal avascular zone are also important parameters on OCTA. Please also investigate the repeatability of FAZ metrics.

: Thank you for your suggestion. We also think that FAZ metrics are important in evaluating RVO. However, we had to exclude 20 eyes because Angioplex software failed to detect FAZ. And among the 37 eyes which Angioplex drew the FAZ line automatically, 13 eyes had inappropriate FAZ line that was either too small or away from the fovea. We drew the conclusion that automated FAZ analysis is unreliable when analyzing eye with RVO. We added in the result (Page 14, line 152-156) and also in discussion (Page 16-17, line 250-257).

4. The study subjects are mixed of CRVO and BRVO. There are also different region of BRVO. It would be interesting to investigate whether the repeatability is different between the region involved and not involved.

: Thank you for your suggestion. We reviewed the OCTA images and metrics of 35 eyes with BRVO. We compared the OCTA images of the regions that were involved and not involved. However, there was no definite difference between the two group. In our study 26 eyes(74%) had macular edma and in most of the eyes with macular edema, all of the ETDRS inner circle area, which we analyzed in this study, were affected regardless of the BRVO region. Therefore the analysis according to the BRVO region was limited. We discussed the following matter in the discussions. (Page 16, line 243-250)

Reviewer #3: The authors present an assessment of OCTA repeatability in retinal vein occlusion.

The authors also report a linear regression analysis of clinical and anatomical parameters affecting VD repeatability and demonstrate how the increase of macular volume reduce the accuracy of the examination.

Our group recently published an angio-OCT study to evaluate Papillary Vessel Density Changes after Intravitreal Anti-VEGF Injections in Central Retinal Vein Occlusion (J Clin Med. 2019 Oct 6;8(10). pii: E1636. doi: 10.3390/jcm8101636). We believe that the peripapillary area might be a region of interest to study microvascular changes when significant macular edema is present. I think it would be interesting to add a comment on this subject.

: Thank you for your thoughtful suggestion.When macular metrics are unreliable due to macular edema, peripapillary metrics can be more reliable and significant data to investigate retinal microvascular changes as you pointed out. We have added the comment in our discussion. (Page 15-16, line 229-232)

In the conclusions section it would be more appropriate to specify that there is a correlation between the entity of macular edema and the repeatability of VD measurement rather than stating that the repeatability is "poor when eyes were associated with macular edema".

: Thank you for your suggestion. We have changed the expression in the conclusions section. ‘Repeatability of VD measurement was significantly affected by central macular thickness.’ ( Page 3, line 46-47)

Minor corrections:

Page 13 line line 120: typos thickness

Page 15 line 220 lower instead of low

: Thank you for your comment. We have checked and corrected the typos.

Submitted filename: Response to the reviewer 0515.docx

Click here for additional data file.

5 Jun 2020

Repeatability of measuring the vessel density in patients with retinal vein occlusion: an optical coherence tomography angiography study.

PONE-D-20-06442R1

Dear Dr. Kim,

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: (No Response)

Reviewer #3: The authors have addressed the comments. The English appears correct. The conclusion is now more in accordance with the findings of the study. I think the study provides interesting data on the repeatability of OCTA assessment in patients with macular edema correlated to RVO.

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Reviewer #2: No

Reviewer #3: No

12 Jun 2020

PONE-D-20-06442R1

Repeatability of measuring the vessel density in patients with retinal vein occlusion: an optical coherence tomography angiography study.

Dear Dr. Kim:

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