October 2019
Volume 60, Issue 13
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Retina  |   October 2019
Analysis of Hyperreflective Dots Within the Central Fovea in Healthy Eyes Using En Face Optical Coherence Tomography
Author Affiliations & Notes
  • Giulia Corradetti
    Retinal Disorders and Ophthalmic Genetics Division, Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States
  • Adrian Au
    Retinal Disorders and Ophthalmic Genetics Division, Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States
  • Enrico Borrelli
    Ophthalmology Department, San Raffaele University Hospital, Milan, Italy
  • Xiaoyu Xu
    Vitreous, Retina, Macula Consultants of New York, New York, New York, United States
  • K. Bailey Freund
    Vitreous, Retina, Macula Consultants of New York, New York, New York, United States
    LuEsther T, Mertz Retinal Research Center, Manhattan Eye, Ear and Throat Hospital, New York, New York, United States
    Department of Ophthalmology, New York University School of Medicine, New York, New York, United States
  • David Sarraf
    Retinal Disorders and Ophthalmic Genetics Division, Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States
    Greater Los Angeles VA Healthcare Center, Los Angeles, California, United States
  • Correspondence: David Sarraf, Retinal Disorders and Ophthalmic Genetics Division, Stein Eye Institute, University of California Los Angeles, 100 Stein Plaza, Los Angeles, CA 90095, USA; sarraf@jsei.ucla.edu
Investigative Ophthalmology & Visual Science October 2019, Vol.60, 4451-4461. doi:https://doi.org/10.1167/iovs.19-27476
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      Giulia Corradetti, Adrian Au, Enrico Borrelli, Xiaoyu Xu, K. Bailey Freund, David Sarraf; Analysis of Hyperreflective Dots Within the Central Fovea in Healthy Eyes Using En Face Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2019;60(13):4451-4461. doi: https://doi.org/10.1167/iovs.19-27476.

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

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Abstract

Purpose: The purpose of this retrospective study was to describe and quantify superficial hyperreflective dots within the central fovea and correlate them with age, using en face and cross-sectional B-scan optical coherence tomography (OCT).

Methods: Healthy eyes, evaluated with a spectral domain instrument (primary cohort) at the Stein Eye Institute (UCLA) and with a swept source instrument (secondary cohort) at the Vitreous Retina Macula Consultants of New York, were included in this study. En face OCT images (3 × 3 mm) segmented at the level of the superior vascular plexus were acquired, and hyperreflective dots in the foveal avascular zone were quantified by two different methodologies. The threshold reflectivity methodology quantified these dots on a cropped en face OCT image using ellipsoid zone mean reflectivity as the threshold cutoff. The OCT B-scan methodology consisted of a manual count of elevated hyperreflective signals on B-scans that colocalized with the dots by en face OCT. Primary outcome was to quantify these dots and correlate them with age.

Results: A total of 44 healthy eyes were evaluated in the primary cohort, and 16 healthy eyes were evaluated in the secondary cohort. The hyperreflective dots steadily increased in number, especially in patients older than 50 years of age, with a strongly positive statistical significant correlation, using both quantitative strategies.

Conclusions: Remarkable superficial hyperreflective dots in the central fovea of healthy subjects are novel anatomical findings that may be readily identified with both en face and cross-sectional OCT and steadily increase in number with age. We propose that these dots may represent a normal anatomical landmark, such as Müller cell end feet or inner limiting membrane basal lamina.

Optical coherence tomography (OCT) imaging has dramatically improved our insight and understanding of the normal and diseased anatomy of the macula.1 This technology can generate cross-sectional images of retinal architecture, similar to histology, in real time and noninvasively, enabling diagnosis of retinal pathology at early stages.25 Cross-sectional spectral domain OCT (SD-OCT), and more recently en face OCT, have revolutionized the evaluation of macular disorders by providing depth-resolved microscopic visualization of the retinal layers.6 With high-density volumetric OCT analysis, the aggregate of B-scans can be reproduced in the coronal or en face plane at a designated segmentation level, which can reveal important information regarding the pattern of distribution of novel anatomical landmarks and clinical abnormalities.7 For example, en face OCT imaging has recently elucidated the pattern of paracentral acute middle maculopathy lesions and the pathogenesis of the related ischemic cascade.8,9 
Using en face OCT analysis in the routine evaluation of patients, we have recently identified a unique and novel anatomical feature remarkable for an aggregation of hyperreflective dots within the central fovea. We sought to determine the anatomical correlate of these dots given their significant location within the visual axis. The purpose of this study was to describe and quantify the hyperreflective dots localized within the foveal avascular zone (FAZ) in healthy subjects according to age. 
Methods
This study was approved by the Institutional Review Board of the University of California – Los Angeles (UCLA) and the Western Institutional Review Board (Olympia, WA, USA), was performed in accordance with the Health Insurance Portability and Accountability Act, and adhered to the principles of the Declaration of Helsinki. Informed consent was obtained from each subject prior to the completion of OCT angiography (OCTA) imaging. 
Study Cohort With Inclusion and Exclusion Criteria
This was a retrospective cross-sectional study of normal eyes (primary cohort) evaluated with OCTA (Optovue, Fremont, CA, USA) at the Stein Eye Institute between 2016 and 2018. The OCTA database was searched, normal eyes were captured, and en face OCT images were evaluated for the characteristic hyperreflective dots within the central fovea in the primary cohort by independent graders (GC and AA). Patients were divided into nine groups (groups 0–8) based on the age decade of life. 
In order to rule out machine-dependent artifacts and to assess the hyperreflective dots using a swept source OCT instrument, we included in our study an additional secondary cohort of healthy eyes evaluated at the Vitreous Retina Macula Consultants of New York and imaged with the (PLEX Elite 9000; Carl Zeiss Meditec, Inc., Dublin, CA, USA). The PLEX Elite software, operating with a central wavelength of 1060 nm, provides a greater axial resolution in tissue. In this secondary cohort, also evaluated according to the age decade of life, the hyperreflective dots within the central fovea on en face OCT were evaluated by only one grader (XX). 
Inclusion criteria included normal eyes that displayed the characteristic hyperreflective dots within the FAZ with high-quality en face OCT imaging, including a signal strength index of at least 60 or greater and quality index of 7 or greater segmented at the level of the superficial vascular plexus (SVC). Eyes with any prior surgery (excluding cataract surgery greater than 6 months prior to enrollment), large refractive error (myopia or hyperopia greater than 3 diopters or astigmatism greater than 2.5 diopters), significant ocular media opacity, or any evidence of vitreoretinal or macular disease were excluded. Patients with ocular media opacity or retinal or vitreoretinal diseases in the fellow eyes were also excluded. En face images with significant motion or segmentation artifacts were also excluded. If both eyes were eligible for enrollment, only one eye, preferentially the right eye, was selected for analysis, based on the quality scan criteria. 
Imaging Protocol and Segmentation
En face OCT imaging was performed with an SD-OCT device (RTVue XR Avanti with AngioVue Software; Optovue) in the primary study group and with the PLEX Elite in the secondary study group. OCT and OCTA images were acquired and analyzed with the 3 × 3-mm scan protocol only. The FAZ was used to define the outer borders in which the hyperreflective dots could be identified and quantified. The FAZ on OCTA was determined using manufacturer-recommended default settings. 
In both the primary and secondary study groups, the structural en face OCT corresponding to the default “superficial” slab segmentation was isolated and analyzed for the hyperreflective dots. The superficial slab was obtained by using the default software segmentation along the SVC and defined by an inner and outer boundary (Fig. 1). Specifically, the inner boundary was positioned 3 μm beneath the internal limiting membrane (ILM), whereas the outer boundary was localized 15 μm beneath the inner plexiform layer (IPL). The two boundaries corresponded to the central foveal excavation and the foveal pit floor. This default slab segmentation was chosen in order to reduce the light reflex signal within the FAZ with en face OCT, which can mask the structural hyperreflective dots (Fig. 2). 
Figure 1
 
En face OCT and corresponding B-scan segmented at the level of the SVC illustrating remarkable hyperreflective dots. Segmentation was 15 μm wide, and boundaries were defined by the inner limiting membrane (ILM) and inner plexiform layer (IPL) aligned along the central foveal depression. (A) The en face OCT image illustrates the presence of hyperreflective dots within the FAZ, corresponding to the foveal pit floor with the OCT B-scan (B).
Figure 1
 
En face OCT and corresponding B-scan segmented at the level of the SVC illustrating remarkable hyperreflective dots. Segmentation was 15 μm wide, and boundaries were defined by the inner limiting membrane (ILM) and inner plexiform layer (IPL) aligned along the central foveal depression. (A) The en face OCT image illustrates the presence of hyperreflective dots within the FAZ, corresponding to the foveal pit floor with the OCT B-scan (B).
Figure 2
 
En face OCT illustrates the light reflex artifact (arrow) within the FAZ, which can be confused with the hyperreflective dots. In our study, we used the default superficial segmentation to neutralize this artifact.
Figure 2
 
En face OCT illustrates the light reflex artifact (arrow) within the FAZ, which can be confused with the hyperreflective dots. In our study, we used the default superficial segmentation to neutralize this artifact.
Methods of Quantification of Hyperreflective Dots
The hyperreflective dots included in the count analysis were described as hyperreflective round- or diamond-shaped dots within the FAZ by en face OCT segmenting along the SVC, corresponding with a distinct hyperreflective signal on OCT B-scans at the level of the floor of the foveal pit. 
In both the primary and secondary cohorts, the number of hyperreflective dots was determined using two methodologies to ensure reproducibility: (1) threshold reflectivity strategy using en face OCT and ImageJ analysis and (2) OCT B-scan colocalization strategy. 
Threshold Reflectivity Strategy Using En Face OCT
The threshold reflectivity strategy using en face OCT was performed on exported and cropped structural en face OCT images. Cropping of the images was determined by the borders of the FAZ, which was isolated by the manufacturer's default settings. This exercise was essential to reduce any confounding signals during image analysis with ImageJ software (1.51m9; http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).1012 The obtained cropped image was binarized and thresholded to quantify the hyperreflective dots by ImageJ software. The hyperreflectivity of the dots was thresholded based on the B-scan ellipsoid zone (EZ) reflectivity.13 
The B-scan EZ, defined as a slab of 21-μm thickness with an inner boundary located 45 μm above the RPE reference, was exported and its mean reflectivity measured. Given that each pixel in the 8-bit exported image ranged from 0 to 255 in the gray scale (0 = maximum hyporeflectivity and 255 = maximum hyperreflectivity), the mean brightness of the B-scan EZ was calculated as the mean of all the pixel values within a square of interest (0.001 mm2 rectangular area) averaged in the central fovea and in the temporal and nasal macula in order to obtain mean values of B-scan EZ brightness not biased by the anatomical location. 
The mean B-scan EZ brightness was used as threshold cutoff value to measure the number of the hyperreflective dots within the FAZ in the cropped en face OCT image using ImageJ. Thresholded images were then used to determine the number and size (mean length) of hyperreflective dots within the FAZ on the cropped 3 × 3-mm en face OCT images (Fig. 3). 
Figure 3
 
Presentation of the 3 × 3-mm en face OCTA and en face OCT images and the cropped, binarized, and thresholded en face OCT images from Image J in order to illustrate the threshold methodology to quantify the hyperreflective dots. (Top left) En face OCTA image segmented at the SVC illustrating the FAZ). The FAZ module is overlayed with the en face OCT to determine the location of the FAZ and then subsequently cropped. (Top right) En face OCT (segmented at the level of the SVC) illustrates the presence of the hyperreflective dots within the overlaid FAZ. (Bottom left) Cropped en face OCT images were adjusted to incorporate the predetermined FAZ and then exported to ImageJ for analysis. (Bottom middle) All en face OCT images were binarized, thresholded, and then analyzed to determine the number of hyperreflective dots using ImageJ. (Bottom right) Thresholded en face OCT image. Mean EZ reflectivity measured on B-scan OCT was used as the threshold. The black spots represent the corresponding hyperreflective dots on en face OCT scans.
Figure 3
 
Presentation of the 3 × 3-mm en face OCTA and en face OCT images and the cropped, binarized, and thresholded en face OCT images from Image J in order to illustrate the threshold methodology to quantify the hyperreflective dots. (Top left) En face OCTA image segmented at the SVC illustrating the FAZ). The FAZ module is overlayed with the en face OCT to determine the location of the FAZ and then subsequently cropped. (Top right) En face OCT (segmented at the level of the SVC) illustrates the presence of the hyperreflective dots within the overlaid FAZ. (Bottom left) Cropped en face OCT images were adjusted to incorporate the predetermined FAZ and then exported to ImageJ for analysis. (Bottom middle) All en face OCT images were binarized, thresholded, and then analyzed to determine the number of hyperreflective dots using ImageJ. (Bottom right) Thresholded en face OCT image. Mean EZ reflectivity measured on B-scan OCT was used as the threshold. The black spots represent the corresponding hyperreflective dots on en face OCT scans.
OCT B-Scan Colocalization Strategy
The OCT B-scan colocalization strategy consisted of manually counting only those hyperreflective dots within the FAZ on 3 × 3-mm en face OCT images that colocalized as distinct hyperreflective signals extending above the ILM on cross-sectional B-scans (Fig. 4). 
Figure 4
 
Patterns of hyperreflective dots with the en face OCT versus the OCT B-scan analysis. En face OCT images segmented at the superficial vascular complex illustrate a cluster of relatively uniform hyperreflective dots in the FAZ (A, B). OCT B-scans, however, display two different morphologies of the hyperreflective dots in the FAZ: an elevated morphology (C) versus a flattened linear morphology (D).
Figure 4
 
Patterns of hyperreflective dots with the en face OCT versus the OCT B-scan analysis. En face OCT images segmented at the superficial vascular complex illustrate a cluster of relatively uniform hyperreflective dots in the FAZ (A, B). OCT B-scans, however, display two different morphologies of the hyperreflective dots in the FAZ: an elevated morphology (C) versus a flattened linear morphology (D).
Only the en face hyperreflective dots that colocalized on the cross-sectional B-scan were included in the counting analysis. The manual count was performed in the primary cohort by independent graders (GC and AA) and in the secondary cohort by one grader (XX). 
Statistical Evaluation
All statistical analyses were performed using statistical software (SPSS Statistics v 21.0; IBM Corp., New York, NY, USA). Age data are expressed as mean ± standard deviation (SD). 
The Bland-Altman plot, Spearman's ρ, and t-test analyses were used to assess the agreement between the threshold reflectivity methodology and the OCT B-scan colocalization methodology. Kruskal-Wallis analysis of variance was used to assess the differences among groups in the quantitative analysis of the size and number of the hyperreflective dots. Multiple comparisons between groups was evaluated using t-test analysis. In these cases, Bonferroni correction was employed with P values < 0.05/36. Otherwise, P value < 0.05 was considered statistically significant. Cohen's κ statistic was determined to evaluate intergrader agreement in the primary cohort. 
Results
Forty-four healthy eyes of 44 patients (26 males and 18 females) were imaged and included in the primary cohort, and 16 additional healthy eyes of 16 patients (6 males and 10 females) were included in a secondary cohort. Average age was 45.2 ± 25.6 years (range, 4–85 years) and 40 ± 20.1 years (range, 18–83 years) for the primary and secondary cohort, respectively. The results are summarized toward the end of this section. 
In the primary cohort, patients were divided into nine different groups according to decade of age (group 0: 0–9 years of age; group 1: 10–19 years of age; group 2: 20–29 years of age; group 3: 30–39 years of age; group 4: 40–49 years of age; group 5: 50–59 years of age; group 6: 60–69 years of age; group 7: 70–79 years of age; group 8: 80–89 years of age). Average Snellen visual acuity (VA) was logMAR 0.11 ± SD 0.08 or 20/25 (Snellen best corrected VA [BCVA] ranged from 20/20 to 20/32) and logMAR 0.1 ± SD 0.05 or 20/25 (Snellen BCVA ranged from 20/20 to 20/25) for the primary cohort and the secondary cohort, respectively. 
Table 1 displays the comparative numerical counts of hyperreflective dots using the en face OCT (threshold reflectivity) methodology versus the OCT B-scan colocalization methodology in the spectral domain cohort. There was no statistically significant difference between these two strategies (P = 0.43), although the OCT B-scan colocalization methodology recorded a slightly lower number of hyperreflective dots, likely due to increased selection stringency. The κ statistic for intergrader agreement for the threshold reflectivity methodology was 0.77 and for the OCT-scan colocalization methodology was 0.95. The Bland-Altman plot analysis illustrated a bias of +2.556 and a 95% limit of agreement between −10.53 and +15.64, indicating a small discrepancy in the measurement of the hyperreflective dots between the two methodologies. The en face OCT threshold reflectivity strategy overestimated the count of the hyperreflective dots by two, on average, compared to the OCT B-scan colocalization strategy. The 95% confidence interval for the bias was 0.5 to 3.6, indicating a small discrepancy between the two methodologies. 
Table 1
 
Quantitative Data of Hyperreflective Dots (as Correlated With Age) Using the En Face OCT Threshold Reflectivity Methodology Versus the Cross-Sectional OCT B-Scan Methodology in the Primary Cohort
Table 1
 
Quantitative Data of Hyperreflective Dots (as Correlated With Age) Using the En Face OCT Threshold Reflectivity Methodology Versus the Cross-Sectional OCT B-Scan Methodology in the Primary Cohort
The Bland-Altman plot showed a small difference between the average number of hyperreflective dots between the two measurement methodologies (Fig. 5) in the primary cohort. Spearman's ρ (Sr) demonstrated a strong statistically significant correlation of +0.97 (P < 0.05) between the two strategies. 
Figure 5
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the primary cohort. A Bland-Altman plot was used to correlate the mean averages and mean differences in the number of hyperreflective dots using two different strategies: en face OCT threshold reflectivity and OCT B-scan colocalization. The plot shows a bias of +2.556 and a 95% limit of agreement between −10.53 and +15.64, indicating a small difference between the two strategies. Bland-Altman plot shows that the en face OCT threshold methodology overestimated the number of hyperreflective dots by two compared to the OCT B-scan colocalization strategy. Bland-Altman findings were confirmed by Spearman's ρ (Sr = +0.97, P < 0.05), indicating a highly statistically significant correlation between the two different methodologies.
Figure 5
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the primary cohort. A Bland-Altman plot was used to correlate the mean averages and mean differences in the number of hyperreflective dots using two different strategies: en face OCT threshold reflectivity and OCT B-scan colocalization. The plot shows a bias of +2.556 and a 95% limit of agreement between −10.53 and +15.64, indicating a small difference between the two strategies. Bland-Altman plot shows that the en face OCT threshold methodology overestimated the number of hyperreflective dots by two compared to the OCT B-scan colocalization strategy. Bland-Altman findings were confirmed by Spearman's ρ (Sr = +0.97, P < 0.05), indicating a highly statistically significant correlation between the two different methodologies.
A positive exponential correlation of hyperreflective dots with age (r = 0.69 for the threshold reflectivity strategy and r = 0.68 for the OCT B-scan colocalization strategy; Fig. 6) was noted. Between age groups, a statistically significant difference in both methodologies (P = 0.0001) was recorded. Notably, hyperreflective dots were identified at an early age (group 1: 2 ± SD 1.75 by thresholding and 1.33 ± SD 0.51 by B-scan count), significantly increased by approximately age 50 (group 5: 9.6 ± SD 8.84) and peaked by age 70 (group 7: 49.4 ± SD 27.66). Subanalysis demonstrated a significant increase in dots in groups 6, 7, and 8 compared to groups 0 and 1 after Bonferroni correction (P = 0.0014). The increase in the number of the hyperreflective dots according to age is represented by the graph in Figure 6 and the collage in Figure 7
Figure 6
 
Graph correlating the number of dots (using the en face threshold methodology and OCT B-scan methodology) with age. Overall, there is an exponential increase in the number of hyperreflective dots with age using both strategies, with a positive correlation coefficient (R = 69% for the threshold methodology; R = 68% for B-scan methodology). Expon., exponential.
Figure 6
 
Graph correlating the number of dots (using the en face threshold methodology and OCT B-scan methodology) with age. Overall, there is an exponential increase in the number of hyperreflective dots with age using both strategies, with a positive correlation coefficient (R = 69% for the threshold methodology; R = 68% for B-scan methodology). Expon., exponential.
Figure 7
 
En face OCT images and registered OCT B-scans from four different age groups. The figures illustrate an increase in the number of the hyperreflective dots according to age as identified with the thresholded en face OCT images (A) and an increase in the corresponding number of lesions as identified with the registered OCT B-scans (B). (A, B) A 4-year-old patient from group 0. En face OCT illustrates one single hyperreflective dot that corresponds to a flattened hyperreflective signal on the registered B-scan OCT. (C, D) A 21-year-old female from group 2. Note there is minimal increase in the number of hyperreflective dots. (E, F) A 55-year-old male from group 5. Note the significant increased number of hyperreflective dots illustrated with both the thresholded en face OCT and the OCT B-scan in this patient from the older cohort. (G, H) A further increase in the number of hyperreflective dots is noted with the en face and OCT B-scan in this 85-year-old patient.
Figure 7
 
En face OCT images and registered OCT B-scans from four different age groups. The figures illustrate an increase in the number of the hyperreflective dots according to age as identified with the thresholded en face OCT images (A) and an increase in the corresponding number of lesions as identified with the registered OCT B-scans (B). (A, B) A 4-year-old patient from group 0. En face OCT illustrates one single hyperreflective dot that corresponds to a flattened hyperreflective signal on the registered B-scan OCT. (C, D) A 21-year-old female from group 2. Note there is minimal increase in the number of hyperreflective dots. (E, F) A 55-year-old male from group 5. Note the significant increased number of hyperreflective dots illustrated with both the thresholded en face OCT and the OCT B-scan in this patient from the older cohort. (G, H) A further increase in the number of hyperreflective dots is noted with the en face and OCT B-scan in this 85-year-old patient.
The B-scan analysis illustrated two different morphologies of the hyperreflective dots, including a flat hyperreflective linear signal or a slightly elevated hyperreflective signal, as illustrated in Figure 4. The different morphology on B-scan was not associated with any other features and did not show any differences between age groups (Table 2; P = 0.43). Additionally, the appearance of the dots on en face OCT did not show any changes with OCT directionality. 
Table 2
 
Average Length (Diameter) of Hyperreflective Dots Versus Age
Table 2
 
Average Length (Diameter) of Hyperreflective Dots Versus Age
The analysis of the data according to age in the secondary cohort using a swept source OCT device (PLEX Elite 9000) in an additional 16 patients illustrated a similar trend (Fig. 8). The Bland-Altman analysis again showed a strong agreement between the two methodologies to quantify the hyperreflective dots using the PLEX Elite 9000 instrument (bias = 1.3, P < 0.05; Fig. 9). The 95% confidence interval was 0.16 to 2.4, showing a small discrepancy between the two methodologies. Bland-Altman findings were confirmed by Spearman's ρ (Sr = + 0.96, P < 0.05), indicating a highly statistically significant correlation between the two different methodologies. 
Figure 8
 
Graph correlating the number of dots (using the threshold methodology and the B-scan methodology) with age in the secondary cohort, that is, the PLEX Elite series. A total of 16 normal eyes were analyzed in this secondary cohort, using a swept source instrument (PLEX Elite 9000) in order to validate the findings in the primary study group of 44 eyes imaged with a different spectral domain system (Angiovue) and to rule out an artifact as an explanation for the hyperreflective dots. A consistent trend is illustrated. The graph displays an increase in the number of hyperreflective dots as correlated with age with both methodologies. The progression appears to exhibit a plateau in the curve and to show a major increase after the age of 55 years, as with the primary cohort.
Figure 8
 
Graph correlating the number of dots (using the threshold methodology and the B-scan methodology) with age in the secondary cohort, that is, the PLEX Elite series. A total of 16 normal eyes were analyzed in this secondary cohort, using a swept source instrument (PLEX Elite 9000) in order to validate the findings in the primary study group of 44 eyes imaged with a different spectral domain system (Angiovue) and to rule out an artifact as an explanation for the hyperreflective dots. A consistent trend is illustrated. The graph displays an increase in the number of hyperreflective dots as correlated with age with both methodologies. The progression appears to exhibit a plateau in the curve and to show a major increase after the age of 55 years, as with the primary cohort.
Figure 9
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the secondary cohort. The Bland-Altman analysis shows a strong positive statistical significant agreement between the two methodologies to quantify the hyperreflective dots, using the PLEX Elite 9000 instrument (bias = 1.3, P < 0.05; Spearman's ρ = +0.96).
Figure 9
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the secondary cohort. The Bland-Altman analysis shows a strong positive statistical significant agreement between the two methodologies to quantify the hyperreflective dots, using the PLEX Elite 9000 instrument (bias = 1.3, P < 0.05; Spearman's ρ = +0.96).
A significant increase in the number of hyperreflective dots was again noted according to age in the secondary cohort, reaching a maximum at the age of 65 years and leveling out at the seventh and eighth decade of life. There was no statistically significant difference in counts between the en face OCT threshold reflectivity versus the OCT B-scan colocalization methodologies (P = 0.5) in the secondary cohort. The variability between patients imaged with the Optovue RTVue XR Avanti versus the PLEX Elite 9000 was not statistically significant by either the threshold reflectivity methodology (F = 0.3, P = 0.84; Table 3) or the OCT B-scan colocalization methodology (F = 0.2, P = 1.3; Table 3). 
Table 3
 
Demographic and Clinical Data of Patients With Hyperreflective Dots in the PLEX Elite 9000 Cohort and Comparative Statistical Analysis of PLEX Elite Cohort (i.e., Secondary Cohort) Versus the Optovue Spectral Domain Group (i.e., Primary Cohort)
Table 3
 
Demographic and Clinical Data of Patients With Hyperreflective Dots in the PLEX Elite 9000 Cohort and Comparative Statistical Analysis of PLEX Elite Cohort (i.e., Secondary Cohort) Versus the Optovue Spectral Domain Group (i.e., Primary Cohort)
In addition, the mean length of the hyperreflective dots was measured with the Optovue RTVue XR Avanti system, and an average length 12.5 ± SD 2.7 μm was noted. A correlation with age was not present, and there were no statistically significant differences in the size of the dots among the nine groups (P > 0.05). 
Discussion
The purpose of the present study was to describe, quantify, and characterize, we believe for the first time, superficial hyperreflective dots detected by en face OCT and cross-sectional OCT B-scan analysis. These hyperreflective dots are a normal OCT finding and are easily differentiated from the larger, pathological, hyperreflective intraretinal foci that can be a biomarker of macular disease in age-related macular degeneration, diabetic retinopathy, and other disorders.9,14 In our study, these remarkable dots were located at the level of the ILM on the floor of the foveal pit and measured an average length of 12.5 ± SD 2.7 μm, increasing in number after the fifth decade of life. We speculate that these en face OCT findings may represent a normal anatomical landmark, either Müller cell end feet and their associated projections to the ILM or the connective fibrils that form the basal lamina of the ILM. 
The architecture of the foveal floor is made up of Müller cell end feet, ILM, and vitreous components. The retinal cells that compose the foveal floor are a subtype of specialized Müller cells and constitute the inner portion of the Müller cell cone. This was first demonstrated by Yamada,15 who displayed the ultraspecialized Müller cells by electron microscopy in an inverted cone bouquet in the floor of the foveal pit. Gass16 suggested that these Müller cells specifically provide structural stability to the fovea, which was confirmed by Syrbe et al.,17 who illustrated that the cell processes and end feet of these ultraspecialized Müller cells create the innermost portion of the foveal pit floor.1821 According to Franze et al.,22 the measured diameter of Müller cell end feet in the vertebrate retina is approximately 12 μm, suggesting that these en face OCT hyperreflective dots may represent the funnel-shaped termination of the Müller cell facing the vitreous body, densely packed in a cobblestone pattern at the inner retinal surface and referred to as Müller cell end feet.22 The Müller cell end feet are known to be comprised of a rich population of mitochondria, which may explain the hyperreflective aspect of the dots with OCT imaging.23 
The basal lamina of the ILM is an electron-dense layer that lies immediately over the specialized Müller cells in the central fovea and appears as dark knobs according to an electron microscopy (EM) study of both normal and diseased eyes.17,24,25 Bringmann et al.25 have provided histologic evidence that the basal lamina of the ILM courses along the irregular inner retinal profile of the Müller cell end feet, and therefore it is possible that the superficial hyperreflective dots may actually represent basal lamina material.17,24 Developmentally, the ILM increases in thickness during the first months of life, stabilizes in thickness in the second decade of life, and then maintains its thickness throughout subsequent decades until the aging process causes further thickening in the parafovea and a progressive thinning in the foveola.26,27 This may explain the increase in OCT hyperreflective dots that we have noted with age in the foveola and not in the parafovea. In eyes that have undergone pars plana vitrectomy with incomplete ILM peeling for macular holes, Nakamura et al.28 speculated that there is fibroproliferation of collagen fibrils and the ILM that creates hyperreflective knobs detected with EM.28,29 
Vitreous collagen fibrils adhere to the fovea anterior to the basal lamina of the ILM, and human vitreous hyalocytes in the vitreous cortex in contact with the ILM appear flattened.26,28,30,31 Histopathologic analysis of posterior vitreous detachment (PVD) has indicated that these vitreous collagen fibrils can persist and may form hyperreflective knobs.26 They can appear as disc-shaped collagenous membranes covering the fovea, or may form a ring along the foveal margin, or may develop a cyst-like structure.31,32 In contrast to Nakamura et al.,28 Gandorfer et al.30 attributed the hyperreflective knobs, detected with SD-OCT and identified after incomplete ILM peeling in eyes with macular holes, to remnants of vitreous fibrils adherent to the basal lamina of the ILM and not fibroproliferation of the ILM. In young patients, persistence of the posterior hyaloid or vitreous remnants may explain the presence of hyperreflective dots in the absence of a PVD.31 
Paques et al.32 described Gunn dots using en face adaptive optics and speculated that these either represented Müller cell end feet or vitreous hyalocytes. However, the unique location in the foveola, the larger dimension, the three-dimensional appearance on B-scan, and the absent detection on fundoscopy indicate that the hyperreflective dots identified in our study are not Gunn dots.32 
A bright light reflex off the foveal pit has been described, and this phenomenon may be more evident in eyes with a wider foveal pit.32,33 This is unlikely to explain the multitude of hyperreflective dots especially remarkable in older patients, nor does it explain the progressive systematic increase correlated with age.33,34 Furthermore, the en face hyperreflective dots were also identified with the registered structural OCT B-scan as elevated lesions, often with a distinct shape and morphology, therefore decreasing the likelihood that these dots are simply a foveal light reflex phenomenon. 
In our study, we detected an increase in hyperreflective dots with age, especially after the sixth decade. The exact mechanism and explanation of these remarkable progressive changes remains unclear. One possibility is that the hyperreflective dots represent Müller cell end feet, which secondarily activate and proliferate with age. Several reports in the literature have described various mechanisms, such as retinal injury, tractional stress, and mechanical stretch, that can lead to the activation, proliferation, and migration of Müller cells.3437 These responses correlate with an increase of extracellular signal-regulated kinase, which is a protein downstream in the mitogen-activated protein kinase cascade, regulating Müller cell proliferation.38 According to this hypothesis, an increase in number of the hyperreflective dots with en face OCT may be due to the increased activation and proliferation of Müller cells with age, secondary to either increased mechanical or cellular retinal stress. Additionally, Müller cells are known to be densely associated with macular pigment. A decrease in Müller cells has been closely correlated with a loss in macular pigment in various diseases (e.g., macular telangiectasia type 2).39 Consistent with our results, some studies have demonstrated an increase in foveal pigment values with age,4043 which may validate our hypothesis that these hyperreflective dots represent Müller cell end feet. 
An alternative explanation for the progressive increase in the hyperreflective dots relates to age-related structural changes in the extracellular matrix and in the basal membranes of the ILM.26,27,37,42 This is evident when comparing the biochemical differences between the adult and fetal ILM. In the adult human retina, age-related changes in the ILM generate an irregular profile associated with long indentations in the retinal tissue as noted with EM, which may explain the increased number of hyperreflective dots we identified (according to age) with en face OCT and OCT B-scan analysis.27,44,45 Furthermore, Syrbe et al.17 noted in their histologic study that the basal lamina of the ILM appeared thin in the foveola and thick in the parafovea. This finding may indicate that Müller cell end feet are more easily visible under the basal lamina in the foveola versus the parafovea. 
Limitations of this study included the retrospective design and the limited sample size across several different age groups, although a total of 60 cases were analyzed. Prospective studies may clarify whether there is a true progression in the number of hyperreflective dots over time. The association with PVD and macular disease was not performed in this investigation and awaits further study. While these hyperreflective lesions may represent an artifact or an insignificant light reflex signal, the confirmation of the presence of these dots with two different methodologic strategies, using two different instruments, and the correlation of the number of these dots with age, indicated that these findings likely represent an anatomical foveal landmark not previously described. While the threshold reflectivity strategy may overestimate the number of hyperreflective dots, we validated the thresholding analysis with OCT B-scan colocalization strategy to identify and quantify the dots with more stringent criteria, and this significantly correlated with the thresholded counts. 
The presence of the hyperreflective dots, identified by en face and B-scan OCT analysis, may represent a novel anatomical landmark. The size of the dots, their location in the FAZ in the foveal pit, and their significant increase in number as correlated with age indicate that they may represent Müller cell end feet or the fibrils of the basal lamina of the ILM, or least likely, vitreous hyalocytes over the surface of the ILM. OCT, despite its significant advances, does not have the ultrastructure resolution to differentiate these anatomical structures. Further clinicopathologic studies with OCT may elucidate the anatomical explanation for these remarkable findings and provide further insight into this commonly identified normal anatomical finding. 
Acknowledgments
Supported by Research to Prevent Blindness, Inc., New York, New York (DS) and the Macula Foundation Inc., New York, New York (DS, KBF). 
Disclosure: G. Corradetti, None; A. Au, None; E. Borrelli, None; X. Xu, None; K.B. Freund, Allergan (C), Genentech (C), Genentech/Roche (R), Heidelberg Engineering (C), Novartis (C), Optovue (C), Zeiss (C); D. Sarraf, Amgen (C), Bayer (C, S), Genentech (C, R), Heidelberg Engineering (R), Novartis (S), Optovue (C, R), Regeneron (R), Topcon (R) 
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Figure 1
 
En face OCT and corresponding B-scan segmented at the level of the SVC illustrating remarkable hyperreflective dots. Segmentation was 15 μm wide, and boundaries were defined by the inner limiting membrane (ILM) and inner plexiform layer (IPL) aligned along the central foveal depression. (A) The en face OCT image illustrates the presence of hyperreflective dots within the FAZ, corresponding to the foveal pit floor with the OCT B-scan (B).
Figure 1
 
En face OCT and corresponding B-scan segmented at the level of the SVC illustrating remarkable hyperreflective dots. Segmentation was 15 μm wide, and boundaries were defined by the inner limiting membrane (ILM) and inner plexiform layer (IPL) aligned along the central foveal depression. (A) The en face OCT image illustrates the presence of hyperreflective dots within the FAZ, corresponding to the foveal pit floor with the OCT B-scan (B).
Figure 2
 
En face OCT illustrates the light reflex artifact (arrow) within the FAZ, which can be confused with the hyperreflective dots. In our study, we used the default superficial segmentation to neutralize this artifact.
Figure 2
 
En face OCT illustrates the light reflex artifact (arrow) within the FAZ, which can be confused with the hyperreflective dots. In our study, we used the default superficial segmentation to neutralize this artifact.
Figure 3
 
Presentation of the 3 × 3-mm en face OCTA and en face OCT images and the cropped, binarized, and thresholded en face OCT images from Image J in order to illustrate the threshold methodology to quantify the hyperreflective dots. (Top left) En face OCTA image segmented at the SVC illustrating the FAZ). The FAZ module is overlayed with the en face OCT to determine the location of the FAZ and then subsequently cropped. (Top right) En face OCT (segmented at the level of the SVC) illustrates the presence of the hyperreflective dots within the overlaid FAZ. (Bottom left) Cropped en face OCT images were adjusted to incorporate the predetermined FAZ and then exported to ImageJ for analysis. (Bottom middle) All en face OCT images were binarized, thresholded, and then analyzed to determine the number of hyperreflective dots using ImageJ. (Bottom right) Thresholded en face OCT image. Mean EZ reflectivity measured on B-scan OCT was used as the threshold. The black spots represent the corresponding hyperreflective dots on en face OCT scans.
Figure 3
 
Presentation of the 3 × 3-mm en face OCTA and en face OCT images and the cropped, binarized, and thresholded en face OCT images from Image J in order to illustrate the threshold methodology to quantify the hyperreflective dots. (Top left) En face OCTA image segmented at the SVC illustrating the FAZ). The FAZ module is overlayed with the en face OCT to determine the location of the FAZ and then subsequently cropped. (Top right) En face OCT (segmented at the level of the SVC) illustrates the presence of the hyperreflective dots within the overlaid FAZ. (Bottom left) Cropped en face OCT images were adjusted to incorporate the predetermined FAZ and then exported to ImageJ for analysis. (Bottom middle) All en face OCT images were binarized, thresholded, and then analyzed to determine the number of hyperreflective dots using ImageJ. (Bottom right) Thresholded en face OCT image. Mean EZ reflectivity measured on B-scan OCT was used as the threshold. The black spots represent the corresponding hyperreflective dots on en face OCT scans.
Figure 4
 
Patterns of hyperreflective dots with the en face OCT versus the OCT B-scan analysis. En face OCT images segmented at the superficial vascular complex illustrate a cluster of relatively uniform hyperreflective dots in the FAZ (A, B). OCT B-scans, however, display two different morphologies of the hyperreflective dots in the FAZ: an elevated morphology (C) versus a flattened linear morphology (D).
Figure 4
 
Patterns of hyperreflective dots with the en face OCT versus the OCT B-scan analysis. En face OCT images segmented at the superficial vascular complex illustrate a cluster of relatively uniform hyperreflective dots in the FAZ (A, B). OCT B-scans, however, display two different morphologies of the hyperreflective dots in the FAZ: an elevated morphology (C) versus a flattened linear morphology (D).
Figure 5
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the primary cohort. A Bland-Altman plot was used to correlate the mean averages and mean differences in the number of hyperreflective dots using two different strategies: en face OCT threshold reflectivity and OCT B-scan colocalization. The plot shows a bias of +2.556 and a 95% limit of agreement between −10.53 and +15.64, indicating a small difference between the two strategies. Bland-Altman plot shows that the en face OCT threshold methodology overestimated the number of hyperreflective dots by two compared to the OCT B-scan colocalization strategy. Bland-Altman findings were confirmed by Spearman's ρ (Sr = +0.97, P < 0.05), indicating a highly statistically significant correlation between the two different methodologies.
Figure 5
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the primary cohort. A Bland-Altman plot was used to correlate the mean averages and mean differences in the number of hyperreflective dots using two different strategies: en face OCT threshold reflectivity and OCT B-scan colocalization. The plot shows a bias of +2.556 and a 95% limit of agreement between −10.53 and +15.64, indicating a small difference between the two strategies. Bland-Altman plot shows that the en face OCT threshold methodology overestimated the number of hyperreflective dots by two compared to the OCT B-scan colocalization strategy. Bland-Altman findings were confirmed by Spearman's ρ (Sr = +0.97, P < 0.05), indicating a highly statistically significant correlation between the two different methodologies.
Figure 6
 
Graph correlating the number of dots (using the en face threshold methodology and OCT B-scan methodology) with age. Overall, there is an exponential increase in the number of hyperreflective dots with age using both strategies, with a positive correlation coefficient (R = 69% for the threshold methodology; R = 68% for B-scan methodology). Expon., exponential.
Figure 6
 
Graph correlating the number of dots (using the en face threshold methodology and OCT B-scan methodology) with age. Overall, there is an exponential increase in the number of hyperreflective dots with age using both strategies, with a positive correlation coefficient (R = 69% for the threshold methodology; R = 68% for B-scan methodology). Expon., exponential.
Figure 7
 
En face OCT images and registered OCT B-scans from four different age groups. The figures illustrate an increase in the number of the hyperreflective dots according to age as identified with the thresholded en face OCT images (A) and an increase in the corresponding number of lesions as identified with the registered OCT B-scans (B). (A, B) A 4-year-old patient from group 0. En face OCT illustrates one single hyperreflective dot that corresponds to a flattened hyperreflective signal on the registered B-scan OCT. (C, D) A 21-year-old female from group 2. Note there is minimal increase in the number of hyperreflective dots. (E, F) A 55-year-old male from group 5. Note the significant increased number of hyperreflective dots illustrated with both the thresholded en face OCT and the OCT B-scan in this patient from the older cohort. (G, H) A further increase in the number of hyperreflective dots is noted with the en face and OCT B-scan in this 85-year-old patient.
Figure 7
 
En face OCT images and registered OCT B-scans from four different age groups. The figures illustrate an increase in the number of the hyperreflective dots according to age as identified with the thresholded en face OCT images (A) and an increase in the corresponding number of lesions as identified with the registered OCT B-scans (B). (A, B) A 4-year-old patient from group 0. En face OCT illustrates one single hyperreflective dot that corresponds to a flattened hyperreflective signal on the registered B-scan OCT. (C, D) A 21-year-old female from group 2. Note there is minimal increase in the number of hyperreflective dots. (E, F) A 55-year-old male from group 5. Note the significant increased number of hyperreflective dots illustrated with both the thresholded en face OCT and the OCT B-scan in this patient from the older cohort. (G, H) A further increase in the number of hyperreflective dots is noted with the en face and OCT B-scan in this 85-year-old patient.
Figure 8
 
Graph correlating the number of dots (using the threshold methodology and the B-scan methodology) with age in the secondary cohort, that is, the PLEX Elite series. A total of 16 normal eyes were analyzed in this secondary cohort, using a swept source instrument (PLEX Elite 9000) in order to validate the findings in the primary study group of 44 eyes imaged with a different spectral domain system (Angiovue) and to rule out an artifact as an explanation for the hyperreflective dots. A consistent trend is illustrated. The graph displays an increase in the number of hyperreflective dots as correlated with age with both methodologies. The progression appears to exhibit a plateau in the curve and to show a major increase after the age of 55 years, as with the primary cohort.
Figure 8
 
Graph correlating the number of dots (using the threshold methodology and the B-scan methodology) with age in the secondary cohort, that is, the PLEX Elite series. A total of 16 normal eyes were analyzed in this secondary cohort, using a swept source instrument (PLEX Elite 9000) in order to validate the findings in the primary study group of 44 eyes imaged with a different spectral domain system (Angiovue) and to rule out an artifact as an explanation for the hyperreflective dots. A consistent trend is illustrated. The graph displays an increase in the number of hyperreflective dots as correlated with age with both methodologies. The progression appears to exhibit a plateau in the curve and to show a major increase after the age of 55 years, as with the primary cohort.
Figure 9
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the secondary cohort. The Bland-Altman analysis shows a strong positive statistical significant agreement between the two methodologies to quantify the hyperreflective dots, using the PLEX Elite 9000 instrument (bias = 1.3, P < 0.05; Spearman's ρ = +0.96).
Figure 9
 
Graph illustrating the level of agreement between the count methodology: en face threshold reflectivity methodology versus OCT B-scan colocalization methodology in the secondary cohort. The Bland-Altman analysis shows a strong positive statistical significant agreement between the two methodologies to quantify the hyperreflective dots, using the PLEX Elite 9000 instrument (bias = 1.3, P < 0.05; Spearman's ρ = +0.96).
Table 1
 
Quantitative Data of Hyperreflective Dots (as Correlated With Age) Using the En Face OCT Threshold Reflectivity Methodology Versus the Cross-Sectional OCT B-Scan Methodology in the Primary Cohort
Table 1
 
Quantitative Data of Hyperreflective Dots (as Correlated With Age) Using the En Face OCT Threshold Reflectivity Methodology Versus the Cross-Sectional OCT B-Scan Methodology in the Primary Cohort
Table 2
 
Average Length (Diameter) of Hyperreflective Dots Versus Age
Table 2
 
Average Length (Diameter) of Hyperreflective Dots Versus Age
Table 3
 
Demographic and Clinical Data of Patients With Hyperreflective Dots in the PLEX Elite 9000 Cohort and Comparative Statistical Analysis of PLEX Elite Cohort (i.e., Secondary Cohort) Versus the Optovue Spectral Domain Group (i.e., Primary Cohort)
Table 3
 
Demographic and Clinical Data of Patients With Hyperreflective Dots in the PLEX Elite 9000 Cohort and Comparative Statistical Analysis of PLEX Elite Cohort (i.e., Secondary Cohort) Versus the Optovue Spectral Domain Group (i.e., Primary Cohort)
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