Retina Drusen and Age-Related Scattered Hypofluorescent Spots on Late-Phase Indocyanine Green Angiography, a Candidate Correlate of Lipid Accumulation

Ling Chen, Xiongze Zhang, Miaoling Li, Yuhong Gan, and Feng Wen State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

Correspondence: Feng Wen, State PURPOSE. Age-related scattered hypofluorescent spots on late-phase indocyanine green Key Laboratory of Ophthalmology, angiography (ASHS-LIA) might represent fundus aging and neutral lipid accumulation. The Zhongshan Ophthalmic Center, Sun present study was conducted to determine the association between drusen and ASHS-LIA and Yat-senUniversity,54SouthXianlie to provide further evidence for our hypothesis. Road, Guangzhou 510060, China; [email protected]. METHODS. Patients who underwent indocyanine green angiography (ICGA), fundus Submitted: June 23, 2018 fluorescein angiography (FFA), and spectral-domain optical coherence tomography (SD- Accepted: September 26, 2018 OCT) at the Zhongshan Ophthalmic Center from January 2014 to March 2018 were reviewed. Patients with soft drusen or ASHS-LIA in the fellow eyes were included in this study. Citation: Chen L, Zhang X, Li M, Gan Y, Wen F. Drusen and age-related RESULTS. A total of 345 patients aged 33 to 87 years (mean: 66.1 6 8.4 years) were included in scattered hypofluorescent spots on this study. Results showed that all patients had ASHS-LIA, among them, 70 patients (20.3%) late-phase indocyanine green angiog- had concurrent soft drusen, while 156 patients (45.2%) had concurrent hard drusen. raphy, a candidate correlate of lipid Furthermore, the incidence of soft drusen was 8.9% (10/112), 21.2% (41/193), and 47.5% (19/ accumulation. Invest Ophthalmol Vis 40), respectively, in the different grades of ASHS-LIA and significantly increased with the grade Sci. 2018;59:5237–5245. https:// of ASHS-LIA (all < 0.01). In addition, soft drusen were located inside the ASHS-LIA in all doi.org/10.1167/iovs.18-25124 P cases and both soft drusen and ASHS-LIA presented as hypofluorescence on late-phase ICGA, while hard drusen were located outside the ASHS-LIA and presented as hyperfluorescence on late-phase ICGA.

CONCLUSIONS. Soft drusen might occur in the presence of ASHS-LIA, and were correlated with the grade of ASHS-LIA. Based on the association between drusen and ASHS-LIA and their ICGA characteristics, we hypothesized that ASHS-LIA might be a candidate correlate of lipid accumulation in Bruch’s membrane. Keywords: age-related, hypofluorescent spots, indocyanine green angiography, drusen, lipid accumulation

ge-related macular degeneration (AMD) is a leading cause of multimodal imaging techniques, including color fundus pho- A vision loss in elderly people worldwide.1 The prognosis for tography (FP), fundus fluorescence angiography (FFA), short- patients with advanced AMD is generally not good; therefore, wavelength fundus autofluorescence (SW-FAF), and spectral- early detection and intervention of AMD are very important. domain optical coherence tomography (SD-OCT).6 Drusen are landmark lesions of early AMD. With the develop- To date, what ASHS-LIA represent is still unclear. Neutral ment of multimodal imaging technology, combined with lipid accumulation in Bruch’s membrane (BrM) with aging is histopathological studies, our understanding of drusen and the most likely possibility.6,8–11 Lipid deposition in BrM their clinical significance has become more comprehensive.2 In increases with age, with more in the macular region than in addition, subclinical deposits, including basal linear deposit the periphery,11,12 and gradually forms a ‘‘lipid wall’’ between (BlinD) and basal laminar deposit (BlamD), have been the basal lamina of retinal pigment epithelium (RPE) and the demonstrated as very important early pathological changes of inner collagenous layer of BrM.13–15 Eventually, apolipoprotein AMD.3–5 Nevertheless, they cannot be detected in vivo at component degradation causes particle fusion and the forma- present. tion of the BlinD.16 The BlinD is a thin layer of soft-drusen-like Recently, we reported a kind of special hypofluorescent material consisting primarily of neutral lipids.16,17 Studies have spot on late-phase indocyanine green angiography (ICGA) and shown that the BlinD and soft drusen are specific for early AMD called it age-related scattered hypofluorescent spots on late- and may confer a risk of progression to advanced AMD.3,18 Thus phase ICGA (ASHS-LIA).6 These hypofluorescent spots were far, as a subclinical deposit, the BlinD has not been visualized in first reported by Shiraki et al.7 in 1999. Both studies showed vivo. In the present study, we propose for the first time that that ASHS-LIA were age-related, presented as hypofluorescence ASHS-LIA might represent the BlinD or pre-BlinD (lipid wall). on late-phase ICGA, were mainly located in the macular region, The BlinD is one piece of AMD pathology, and its specific and could be confluent. Furthermore, our study revealed that role in progression beyond being associated with drusen is not both the occurrence and grade of ASHS-LIA increased with age; yet clear. The BlinD and soft drusen are diffuse and focal no corresponding abnormalities were detected by other deposits, respectively, of the same lipoprotein-derived debris,

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FIGURE 1. Multimodal imaging of ASHS-LIA. Normal fundi fellow eye (BCVA: 20/20) from a 61-year-old man; his left eye was diagnosed with PCV (not shown). No corresponding changes were present in the color FP (A), SW-FAF (B), FFA (C), and early-phase ICGA (D). (E) ASHS-LIA (red arrow) were distributed in the macular region on late-phase ICGA, with partial confluence. (F) SD-OCT (the green line in E) showed that the retinal structure was generally normal and that the RPE band was intact and smooth, with no intraretinal, subretinal, or sub-RPE deposition corresponding to ASHS-LIA.

and both are located in precisely the same plane.16,19 The after dye injection), (2) primary distribution in the macular purpose of the current study was to evaluate the association region, (3) regular arrangement pattern with possible conflu- between ASHS-LIA and drusen (especially soft drusen) and to ence, and (4) no corresponding abnormalities on color FP, SW- provide further evidence for our hypothesis that ASHS-LIA FAF, FFA, and SD-OCT (Fig. 1). Furthermore, according to the might represent the BlinD or pre-BlinD (lipid wall). region of involvement, ASHS-LIA were divided into three grades: grade 1, macular region; grade 2, macular region and around the optic disc; and grade 3, throughout the posterior MATERIALS AND METHODS pole. Drusen were determined based on multimodal imaging.20 Hard drusen were defined as discrete yellow-white deposits This is a hospital-based retrospective study. Patients who were situated underneath the RPE with diameters less than 63 lm, referred to the Zhongshan Ophthalmic Center for ICGA, color and more compact structures than soft drusen. Soft drusen FP, FFA, and SD-OCT examination between January 2014 and were defined as mound-like deposits situated between the March 2018 were reviewed. The study was conducted reflective band representing the RPE and its basal lamina and according to the Declaration of Helsinki and was approved the remainder of BrM, with diameters usually ‡125 lm and by the Institutional Review Board of the Zhongshan Ophthal- less well demarcated structures than hard drusen. mic Center. Potential risks associated with the ICGA and FFA Subretinal drusenoid deposits (SDDs) (originally called examination were fully discussed with the patients, and reticular pseudodrusen) also presented as hypofluorescent written informed consent was obtained from all included spots on late-phase ICGA but had different distribution patients. patterns and locations compared with ASHS-LIA, and SDDs To prevent lesions from interfering with the assessment of could be detected via other multimodal imaging techniques, fluorescence, only fellow eyes with normal fundi or with soft including color FP, infrared reflectance, and SD-OCT.21–23 A or hard drusen were screened for the study, although ASHS-LIA striking characteristic of SDDs is that they seem to follow the always appeared in both eyes of any patient. The inclusion topography of rod photoreceptors, starting in the perifovea, criterion was soft drusen or ASHS-LIA in the study eye. And the sweeping around the optic nerve head, and encroaching on right eye was included in the study when both eyes of the patient met the inclusion criteria. Patients with bilateral lesions or inflammatory conditions involving the choroid, such as TABLE 1. The Diagnostic Information and the Demographic Charac- teristics of the Included Patients multifocal choroiditis, and patients who had a previous ophthalmological intervention procedure, such as laser coag- Drusen and ASHS-LIA n Age Male (%) ulation, vitrectomy, anti-VEGF injection, or photodynamic therapy in the study eye, were excluded. Patients with PCV 228 66.4 6 7.7 68.4 evidence of significant cataracts or vitreous opacity, which nAMD 41 66.9 6 8.7 73.2 influences imaging quality and fluorescence judgment, were Dry AMD 17 70.0 6 7.6 52.9 also excluded from the study. After these criteria were applied, CSC 11 60.4 6 9.5 63.6 345 fellow eyes with normal fundi or with drusen were eligible RVO 9 62.6 6 10.5 66.7 for inclusion in this study. IPED 9 67.7 6 5.9 88.9 ASHS-LIA were determined based on the following criteria Other 30 63.4 6 10.4 66.7 6 according to our previous study : (1) the presence of Data represent n, mean 6 SD, or %. Dry AMD, dry age-related hypofluorescent spots on late-phase ICGA (20–40 minutes macular degeneration; CSC, central serous chorioretinopathy.

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FIGURE 2. Multimodal imaging of soft drusen and ASHS-LIA. The right eye (BCVA: 20/50) from a 78-year-old man with PCV in his left eye (not shown). (A) Color FP showed substantial yellow-white drusen-like material deposited in the macular region (yellow arrow). (B) FFA showed slight hyperfluorescence corresponding to the drusen (yellow arrow). (C) Late-phase ICGA showed that the soft drusen presented as dense hypofluorescence in the macular region (yellow arrow), and that ASHS-LIA were present in the macular region and its surround (red arrow). Soft drusen located inside the ASHS-LIA. (D) SD-OCT (the green line in C) showed drusenoid pigment epithelium detachment (yellow arrow) corresponding to the soft drusen. No changes corresponding to ASHS-LIA were observed via color FP, FFA, and SD-OCT.

the fovea only in late stages.24,25 Therefore, SDDs can be easily When the two retina physicians disagreed, discrepancies in differentiated from ASHS-LIA, and patients with SDDs have their findings were referred to a third retina specialist (Feng been excluded as ASHS-LIA in this study. In addition, Wen) for final determination. hypofluorescence on late-phase ICGA could be observed in Statistical analyses were performed with SPSS Version 21.0 patients with disturbance of the choroidal circulation, such as software (SPSS, Inc., Chicago, IL, USA). Descriptive statistics chronic central serous chorioretinopathy (CSC),26–28 and in (means 6 SDs) of normally distributed variables and geometric patients with inflammatory diseases, such as punctate inner means with 95% confidence intervals (CIs) of nonnormally choroidopathy (PIC) and multiple evanescent white dot distributed variables were calculated. Comparisons were 29,30 syndrome (MEWDS), which have also been excluded as evaluated by independent samples t-test, 1-way ANOVA ASHS-LIA in the present study. (normal data) or v2 test (nonnormal data). Significant Demographic information, medical records, and multimodal differences were defined as P values < 0.05. imaging data were reviewed. The best-corrected visual acuity (BCVA) was measured with Snellen charts, and a comprehen- sive ophthalmological examination including slit-lamp exami- RESULTS nations and dilated funduscopy was conducted. Color FP was performed with a Zeiss FF450 plus fundus camera (Carl Zeiss, A total of 345 fellow eyes from 345 patients were included in Inc., Jena, Germany). ICGA, FFA, and SW-FAF were performed this study. The patient ages ranged from 33 to 87 years (mean: with a Heidelberg retina angiogram (Spectralis HRA, Heidel- 66.1 6 8.4 years), and the male to female ratio was 2.2:1. The berg Engineering, Heidelberg, Germany). SD-OCT was per- mean BCVA was a Snellen equivalent of 20/25 (range: 20/16– formed with an HRAþOCT Spectralis (Heidelberg Engineering). 20/50). Table 1 shows the diagnostic information and the Two retina physicians (Ling Chen and Xiongze Zhang) demographic characteristics of the included patients. Among independently evaluated and diagnosed the patients, evaluated the 345 included patients, 228 patients had polypoidal the ASHS-LIA and the grades, and assessed the soft or hard choroidal vasculopathy (PCV); 41 patients had neovascular drusen and the association between ASHS-LIA and drusen. AMD (nAMD); 17 patients had dry AMD; 11 patients had CSC; 9

TABLE 2. ASHS-LIA and Drusen Among Different Age Groups

Age Range, y 31–40 41–50 51–60 61–70 71–80 81–90 Total

n 1 8 72 164 83 17 345 ASHS-LIA, n/% 1/100 8/100 72/100 164/100 83/100 17/100 345/100 SD, n/% 0/0 0/0 0/0 23/14.0 35/42.2 12/70.6 70/20.3 HD, n/% 0/0 0/0 15/20.8 73/44.5 53/63.9 15/88.2 156/45.2 Data represent n or %. SD, soft drusen; HD, hard drusen.

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of them was in the age group of 31 to 40 years. Furthermore, soft drusen were only detected in patients over 60 years of age, including 23 of 164 (14%) patients aged 61 to 70 years, 35 of 83 (42%) patients aged 71 to 80 years, and 12 of 17 (71%) patients aged 81 to 90 years. The incidence of soft drusen increased significantly with age (v2 test, all P < 0.001). Moreover, hard drusen were detected in only patients over 50 years of age, including 15 of 72 (21%) patients aged 51 to 60 years, 73 of 164 (45%) patients aged 61 to 70 years, 53 of 83 (64%) patients aged 71 to 80 years, and 15 of 17 (88%) patients aged 81 to 90 years. The incidence of hard drusen increased significantly with age (v2 test, all P < 0.001). Table 3 shows patients with different grades of ASHS-LIA. Our findings showed that the mean age of patients was significantly increased with the grade of ASHS-LIA (1-way ANOVA, all P < 0.001). Nevertheless, there was no significant correlation between gender and the grade of ASHS-LIA. In addition, 10 of 112(8.9%)patientswithgrade1ASHS-LIA,41of193(21.2%) patients with grade 2 ASHS-LIA, and 19 of 40 (47.5%) patients with grade 3 ASHS-LIA had soft drusen. The incidence of soft drusen was significantly increased with the grade of ASHS-LIA (v2 test, all P < 0.01). Additionally, 38 of 112 (33.9%) patients with grade 1 ASHS-LIA, 86 of 193 (44.6%) patients with grade 2 ASHS- LIA, and 32 of 40 (80%) patients with grade 3 ASHS-LIA had hard drusen. The incidence of hard drusen in patients with grade 3 ASHS-LIA was significantly higher than that in patients with grade 1 and grade 2 ASHS-LIA (both P < 0.001). Figure 1 shows the multimodal imaging of ASHS-LIA. ASHS- FIGURE 3. Multimodal imaging of hard drusen and ASHS-LIA. The right LIA presented as hypofluorescence on late-phase ICGA were eye (BCVA: 20/20) from a 66-year-old woman with PCV in her left eye mainly located in the macular region and could be confluent (not shown). (A) Color FP showed that yellow-white hard drusen were (Fig. 1E, red arrow). No corresponding abnormalities were distributed in the posterior pole (yellow arrow). (B) FFA revealed detected by other multimodal imaging techniques, including numerous hyperfluorescent spots corresponding to hard drusen color FP, SW-FAF, FFA, early-phase ICGA, and SD-OCT. (yellow arrow). (C) Late-phase ICGA showed ASHS-LIA (red arrow) Figure 2 shows a representative example of soft drusen and throughout the posterior pole, with remarkable confluence. Hard drusen presented as hyperfluorescent spots (yellow arrow) and were ASHS-LIA. Both ASHS-LIA (Fig. 2C, red arrow) and soft drusen located outside the ASHS-LIA. (D) ICGA merged image showed the (Fig. 2C, yellow arrow) presented as hypofluorescence on late- distribution of ASHS-LIA and hard drusen. (E) SD-OCT (the white line phase ICGA, and were mainly located in the macular region. in D) showed that no changes corresponding to the ASHS-LIA were Soft drusen was located within the bounds of the region with observed. (F) SD-OCT (the yellow line in D) showed focal high ASHS-LIA in all cases, with lower fluorescence than ASHS-LIA reflective deposits situated underneath the RPE (yellow arrow), on late-phase ICGA. Figure 3 shows a representative example of corresponding to hard drusen. hard drusen and ASHS-LIA. Hard drusen were mainly distribut- ed in the peripheral retina, but there were 32 cases with hard patients had retinal vein obstruction (RVO); 9 patients had drusen distributed in the posterior pole, even in the macular idiopathic pigment epithelium detachment (IPED); and 30 region. Furthermore, hard drusen (Fig. 3C, yellow arrow) patients had other fundus diseases, including pathological presented as hyperfluorescent spots on late-phase ICGA and were always located outside the ASHS-LIA (Fig. 3C, red arrow). myopia, retinal macroaneurysms, epiretinal membrane, focal We also display other conditions with hypofluorescence choroidal atrophy, choroidal hemangioma, Coats’ disease, optic that could be distinguished from ASHS-LIA. Figure 4 shows a neuropathy, and syphilitic retinitis. representative example of hypofluorescence resulting from All 345 patients had ASHS-LIA, among them, 70 patients disturbance of the choroidal circulation in CSC patients. This (20.3%) had concurrent soft drusen, and 156 patients (45.2%) kind of hypofluorescence was caused by focal filling defects in had concurrent hard drusen. Table 2 shows the ASHS-LIA and the choriocapillaris, so it could be an irregular shape and might drusen among the different age groups by decade. The findings occur at any position in the fundus. Moreover, it was better showed that among the 345 patients with ASHS-LIA, only one demarcated than ASHS-LIA and commonly surrounded by

TABLE 3. Demographic Characteristics and Drusen Incidence of Patients With Different Grades of ASHS-LIA

P Values

Grade 1 Grade 2 Grade 3 Grade 1 Grade 2 Grade 1 (n ¼ 112) (n ¼ 193) (n ¼ 40) vs. Grade 2 vs. Grade 3 vs. Grade 3

Age 61.9 6 8.1 67 6 7.2 73.7 6 8.1 P < 0.001 P < 0.001 P < 0.001 Male, % 67.0 69.4 67.5 0.655 0.810 0.951 SD, n (%) 10 (8.9) 41 (21.2) 19 (47.5) 0.005 0.001 P < 0.001 HD, n (%) 38 (33.9) 86 (44.6) 32 (80) 0.068 P < 0.001 P < 0.001 Data represent n, mean 6 SD, or %. Grade 1, ASHS-LIA in the macular region; grade 2, ASHS-LIA in the macular region and around the optic disc; grade 3, ASHS-LIA throughout the posterior pole; SD, soft drusen; HD, hard drusen.

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FIGURE 4. Multimodal imaging of disturbance of choroidal circulation in CSC. The left eye (BCVA: 20/40) from a 50-year-old man; he was diagnosed with central serous chorioretinopathy 2 years ago. (A) Color FP showed irregular lesions and pigment derangement in the macular region. (B)FFA revealed hyperfluorescence due to depigmentation. (C) Early-phase ICGA showed choroidal vascular dilation and hyperpermeability in the macular region. (D) Late-phase ICGA showed irregular hypofluorescence that was well demarcated and surrounded by hyperfluorescent rings. (E) SD-OCT (the green line in D) showed increasing subfoveal choroidal thickness (428 lm) and abnormalities of the ellipsoid zone and RPE.

hyperfluorescent rings (Fig. 4D). Furthermore, abnormalities hypofluorescent spots were uneven in size, were irregular in corresponding to the area of hypofluorescence in this case distribution, and had lower hypofluorescence than ASHS-LIA could be detected by color FP, FFA, early-phase ICGA, and SD- (Fig. 5E). In addition, abnormalities corresponding to the OCT. Figure 5 shows a representative example of hypofluor- hypofluorescent spots could be detected by color FP, SW-FAF, escent spots on late-phase ICGA in patients with PIC. These FFA, early-phase ICGA, and SD-OCT.

FIGURE 5. Multimodal imaging of PIC. The left eye (BCVA: 20/32) from a 28-year-old woman; she was diagnosed with binocular PIC. (A) Color FP showed yellow-white punctate lesions in the macular region. (B) SW-FAF showed hypoautofluorescent spots of PIC (white arrow) and hyperautofluorescent spots of MEWDS (yellow arrow). (C) FFA revealed hyperfluorescent spots of lesion staining. (D) Early-phase ICGA revealed hypofluorescent spots. (E) More hypofluorescent spots were indicated in the late-phase ICGA than in the early-phase ICGA, with inhomogeneous enhancement of background fluorescence. (F) Several active PIC lesions were detected on SD-OCT (the green line in E).

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FIGURE 6. Proposal as to how the BlinD and soft drusen presented as varying degrees of hypofluorescence on late-phase ICGA. (A) Normal structure of the RPE, BrM, and choroidal capillaries (CC). BrM consists of five layers of connective tissue: 1, basal lamina of the RPE (RPE-BL); 2, inner collagenous layer (ICL); 3, elastic layer (EL); 4, outer collagenous layer (OCL); and 5, basal lamina of the choriocapillary endothelium (CC-BL). A small green circle represents indocyanine green (ICG) dye. ICG dye could extravasate into the choroidal stroma, pass through BrM and eventually be taken up by the RPE. (B) Blue material represents the BlamD, and dark yellow material represents the BlinD. The BlinD formed between the ICL of BrM and the RPE basal lamina, containing mainly neutral lipids, could reduce ICG dye through BrM into the RPE. (C) The confluent BlinD further reduced ICG dye through BrM into the RPE. (D) Both soft drusen and the BlinD were located in precisely the same sub-RPE space and contained mainly neutral lipids. Soft drusen could impede ICG dye through BrM into the RPE to a higher degree than the BlinD because of thicker deposits.

DISCUSSION imaging features, we speculated that ASHS-LIA might represent neutral lipids accumulating in BrM.6 BrM is the innermost 2 to In the present study, we evaluated the association between 4 lm of the choroid (subjacent to the RPE), consisting of five drusen and ASHS-LIA and hypothesized that ASHS-LIA might layers of connective tissue.19 Lipoproteins begin to accumulate represent the BlinD or pre-BlinD (lipid wall). Our findings within BrM by the fourth decade of life, slowly increasing and showed that all eyes with soft drusen had concurrent ASHS-LIA eventually leading to the formation of a ‘‘lipid wall,’’ a and that the incidence of soft drusen significantly increased precursor of the BlinD.9,13,15 The BlinD was described as with the grade of ASHS-LIA. Both soft drusen and ASHS-LIA membranous debris in previous studies due to its ultrastruc- presented as hypofluorescence on late-phase ICGA, and soft ture of coiled membranes.5,31,32 However, with lipid-preserv- drusen were located within the bounds of the region with ing ultrastructural techniques, Curcio et al. demonstrated that ASHS-LIA in all cases. In contrast, hard drusen presented as the BlinD was in fact a thin, tightly packed layer (0.4–2 lm) of hyperfluorescence on late-phase ICGA, and were located lipoprotein-derived debris containing neutral lipids.13,33 The outside the region with ASHS-LIA. BlinD and soft drusen are diffuse and focal deposits, Our recent study, with a large sample size and a large age respectively, of the same lipoprotein-derived debris, and both range, demonstrated that the occurrence and grade of ASHS- are located in precisely the same plane.9,16 It has been LIA increased with age.6 Therefore, ASHS-LIA might represent demonstrated that the BlinD and soft drusen are specific for the aging of the fundus. Based on the age correlation, early AMD and may be significantly related to the progression distribution, confluent features, ICGA, and other multimodal to advanced AMD.3,18 Nevertheless, the BlinD, as one piece of

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the retinal and choroidal vessels in the early phase after injection, but it was extravasated into the choroidal stroma and accumulated within the RPE over time.39,40 An in vitro experiment demonstrated that cultured human RPE cells took up ICG dye.41 Recently, Tam et al.42 demonstrated that RPE cells contributed to the fluorescence signal observed in the late phase of ICGA. Both the local and diffusing deposition of neutral lipids between BrM and the RPE (soft drusen and the BlinD) would impede hydrophilic moieties (ICG dye) through BrM into the RPE.43–45 Figures 6 and 7 show the proposal as to how the BlinD and soft drusen can present as varying degrees of hypofluorescence on late-phase ICGA. Under normal conditions, ICG dye could extravasate into the choroidal stroma, pass through BrM, and eventually be taken up by the RPE. Therefore, homogeneous background fluorescence was observed on late-phase ICGA (Figs. 6A, 7A). With age, the BlinD formed between the inner collagenous layer of BrM and the RPE basal lamina, which could reduce ICG dye through BrM into the RPE and present as hypofluorescent spots on late- phase ICGA (Figs. 6B, 7B). When the BlinD became confluent, confluent hypofluorescence was observed on late-phase ICGA (Figs. 6C, 7C). In addition, soft drusen would have even lower hypofluorescence than the BlinD because of thicker deposits of neutral lipids (Figs. 6D, 7D). Additionally, our findings showed that hard drusen were FIGURE 7. Fluorescence characteristics on late-phase ICGA corre- sponding to the proposal. (A) Under normal conditions, ICG dye could always located outside the ASHS-LIA, regardless of the extravasate into the choroidal stroma, pass through BrM and eventually peripheral retina or the macular region. Hard drusen, be taken up by the RPE. Therefore, homogeneous background consisting of homogeneous hyalinized material, manifested fluorescence was observed on late-phase ICGA. (B) With aging, neutral hyperfluorescence on ICGA because the high content of lipids accumulate in BrM, reducing ICG dye through BrM into the RPE. phospholipids binds more ICG dye.34,46 Hard drusen are Hence, hypofluorescent spots were observed in the macular region. considered a normal consequence of age; however, why they (C) Neutral lipids gradually increased and became confluent; hence, are always located outside the ASHS-LIA is still unclear. We confluent hypofluorescence were observed in the macular region. (D) Soft drusen formed in the presence of diffuse deposits of neutral lipids inferred that this might be due to the differences in and contained mainly neutral lipids. Hence, soft drusen presented as composition and polarity between the hard drusen and ASHS- even lower hypofluorescence than ASHS-LIA on late-phase ICGA. LIA material (BlinD), considering that hard drusen, soft drusen, and BlinD were located in the same sub-RPE space.47 This finding provides further evidence for our hypothesis, but further research is needed. AMD pathology, cannot yet be detected in vivo, and thus, its This study has limitations. As a clinic-based retrospective specific role in progression beyond being associated with study, selection bias might occur. No longitudinal observa- drusen is not yet clear. tion was made because ICGA is an invasive examination. In the current study, we reviewed patients who underwent Furthermore, we have no direct clinicopathologic correla- multimodal imaging examination, and we found that all eyes tion evidence, and we cannot explain why ASHS-LIA present with soft drusen had concurrent ASHS-LIA and that the as regular scattered spots before confluence. Nevertheless, incidence of soft drusen increased with the grade of ASHS- previous pathological findings about lipid deposition, the LIA. This means that soft drusen may occur in the presence of BlinD, and soft drusen provided us with a great deal of ASHS-LIA. A previous clinicopathological study demonstrated information.3,8,9,11,13–15,17,37,38 We included a large number that soft drusen were found in only eyes with diffuse deposits of patients who underwent multimodal imaging examina- 32 (BlinD). Therefore, it provided evidence for the correspon- tions. dence between ASHS-LIA and BlinD. Moreover, both soft In conclusion, our study indicated that soft drusen might drusen and ASHS-LIA presented as hypofluorescence on late- occur in the presence of ASHS-LIA and that the incidence of phase ICGA, and soft drusen were located inside the region soft drusen significantly increased with the grade of ASHS-LIA. with ASHS-LIA and had lower fluorescence than ASHS-LIA. As Furthermore, soft drusen were located inside the ASHS-LIA in we know, indocyanine green dye is water soluble and does not all cases, and both the soft drusen and ASHS-LIA presented as bind to neutral lipids, such as esterified and unesterified hypofluorescence on late-phase ICGA. In contrast, hard drusen cholesterol,34 which can explain the hypofluorescence of soft were always located outside the ASHS-LIA and presented as drusen on late-phase ICGA and give us sufficient reason to hyperfluorescence on late-phase ICGA. We hypothesized that speculate that the BlinD, containing the same lipoprotein- ASHS-LIA might be a candidate correlate of lipid accumulation derived debris as in soft drusen, might also present as in BrM, and might represent histopathological BlinD/pre- hypofluorescence on late-phase ICGA.3,35,36 Therefore, the BlinD. BlinD, with similar age correlation, distribution, confluent features, relationship with soft drusen, and possible ICGA Acknowledgments characteristics, is a likely candidate for histologic correlate of ASHS-LIA.5,6,12,17,37,38 Supported by the National Natural Science Foundation of China Then, how did soft drusen and the BlinD (ASHS-LIA) lead to (Grant Number: 81470647). varying degrees of hypofluorescence on late-phase ICGA? A Disclosure: L. Chen, None; X. Zhang, None; M. Li, None; Y. Gan, previous study demonstrated that ICG dye was located within None; F. Wen, None

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