- Email: [email protected]
Appearance of Regressing Drusen on Optical Coherence Tomography in Age-related Macular Degeneration
Appearance of Regressing Drusen on Optical Coherence Tomography in Age-related Macular Degeneration Giuseppe Querques, MD, PhD, Anouk Georges, MD, PhD, Naima Ben Moussa, MD, Margaret Sterkers, MD, Eric H. Souied, MD, PhD Objective: To describe and interpret a multilaminar sub-retinal pigment epithelium (RPE) intense hyperreflectivity observed in vivo in eyes clinically diagnosed with regressing drusen. Design: Observational case series. Participants: Twenty-three consecutive patients clinically diagnosed with regressing calcific drusen due to nonneovascular age-related macular degeneration (AMD). Methods: Patients were submitted to confocal scanning laser ophthalmoscopy (cSLO) fundus imaging and “eye-tracked” spectral-domain optical coherence tomography (SD-OCT). Main Outcome Measures: Localization and possible origin and composition of the multilaminar sub-RPE hyperreflectivity. Results: Thirty eyes of 23 consecutive patients (8 male and 15 female; mean age, 82.710.1 years) showing on SD-OCT an intense multilaminar sub-RPE hyperreflectivity, which matched with regressing calcific drusen as visualized by cSLO infrared (IR) and MultiColor (Heidelberg Engineering, Heidelberg, Germany) images, were included in this study. The multilaminar hyperreflectivity was found to localize to beneath the RPE and above the outer Bruch’s membrane (oBM) layer. A mean of 1.2 multilaminar sub-RPE hyperreflectivities per SD-OCT scan were identified by 2 readers. The SD-OCT analysis allowed the 2 readers to describe 3 different types of sub-RPE hyperreflectivity. “Type 1” laminar/multilaminar hyperreflectivity (found in 24 scans of 12 eyes) was characterized by an intense signal originating from what we interpreted as the inner Bruch’s membrane (iBM) layer. “Type 2” multilaminar hyperreflectivity (found in 130 scans of 27 eyes) was characterized by an intense signal originating from the oBM layer. “Type 3” multilaminar fragmented hyperreflectivity (found in 22 scans of 11 eyes) was characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. Conclusions: We describe a novel SD-OCT finding appearing as multilaminar sub-RPE intense hyperreflectivity observed in vivo in eyes with regressing drusen. This multilaminar sub-RPE hyperreflectivity could be interpreted as layers of lipid mineralization (membranous debris also called “lipoprotein-derived debris” developing calcification), internal and external to the basement membrane, with different degrees of fragmentation. Ophthalmology 2014;121:173-179 ª 2014 by the American Academy of Ophthalmology.
Deposition of extracellular material between the basal lamina of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch’s membrane is generally referred to as “drusen.”1 Drusen usually appear after the age of 50 years and are considered to be the main risk factors of age-related macular degeneration (AMD).2 Large AMD drusen are yellow-white, mound-like RPE elevations typically 63 to 1000 mm in diameter.3 Histopathologic investigations showed that large drusen are mounds external to the RPE basement membrane containing membranous debris (also called “lipoproteinderived debris”), a neutral lipid-rich material.3 Basal linear deposits and large drusen should be regarded as the same lesion.4e6 A basal linear deposit consists of a thin, relatively extensive layer of membranous profiles.7 This is in contradistinction to a basal laminar deposit, a diffusely 2014 by the American Academy of Ophthalmology Published by Elsevier Inc.
deposited material that lies internal to the RPE basement membrane, and is composed of fibrils, amorphous material, and a banded component that is reminiscent of long-spacing collagen.8 Drusen growth is characterized by the continued deposition (overproduction/underclearance) of drusen-associated constituents (membranous debris). Early complexes within Bruch’s membrane, such as immune complexes, or other local ligands might serve as “nucleation sites” for the deposition of additional self-aggregating proteins or lipids.4 Curcio et al9 demonstrated that Folch-extractable lipid, dominated by esterified and unesterified cholesterol, enriches drusen and basal deposits in human eyes with AMD (w40% of drusen content).10 A source for cholesterol in the sub-RPE space may be plasma, endogenous synthesis, ISSN 0161-6420/14/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.06.024
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photoreceptor disk shedding, and assembly of an apolipoprotein B (apoB)-lipoprotein by RPE. In drusen “maturation,” several layers of coiled membranes lay between the RPE and its basement membrane (membranous debris crossing the basal laminar deposit in strands or mounds).5,11,12 These membranous debris extend inward between adjacent RPE cells. A thin stratus of membranes also lay external to the basement membrane. Regressing drusen show evidence of not only reduced formation of membranous debris but also its elimination by macrophages. Material that is not removed develops calcification.5 A better understanding of the physiopathologic features of drusen is required to develop rational and effective AMD therapies. Drusen size, type, and composition reveal both past formative processes4,13,14 and future progression of disease.4,13,14 Because of the paucity of histopathologic information about eyes previously imaged in the clinic, we could refer to contemporary multimodal, high-resolution in vivo imaging to interpret differences in drusen appearance. Spectralis high-resolution spectral-domain (SD) optical coherence tomography (OCT) (Spectralis SD-OCT; Heidelberg Engineering, Heidelberg, Germany) is a high-speed OCT system (up to 40 000 axial scans per second) using SD/ Fourier domain detection, with an optical axial image resolution of 7 mm (digital axial image resolution of 3.5 mm). In Spectralis SD-OCT, confocal scanning laser ophthalmoscopy (cSLO) with real-time eye tracking technology allows one to track the eye and guide OCT to the selected location, thus giving a real-time reference for locating the SD-OCT scan. The purpose of this study was to describe and interpret a multilaminar sub-RPE intense hyperreflectivity observed in vivo in eyes clinically diagnosed with regressing drusen due to nonneovascular AMD.
Methods Twenty-three patients clinically diagnosed with regressing drusen (documented by retrospective evaluation of progression) due to nonneovascular AMD who consecutively presented at the Retinal Service of the University Eye Clinic of Creteil between September and December 2012 were submitted to cSLO fundus imaging and “eye-tracked” SD-OCT. Inclusion criteria were age >50 years; presence on fundus biomicroscopy of soft indistinct drusen combined with RPE changes and regressing calcific drusen (appearing as refractile lesions) in the macular area as manifestation of nonexudative AMD, with no evidence of choroidal neovascularization on fluorescein angiography and indocyanine green angiography; no intraretinal or subretinal fluid; and no sub-RPE/ retinal hemorrhage. Exclusion criteria were signs of any other retinal disease in the study eye, such as retinal vascular disease (i.e., diabetic retinopathy and retinal vein occlusion) or vitreoretinal disease (i.e., vitreomacular traction syndrome and epiretinal membrane; presence of choroidal neovascularization (i.e., exudative AMD; any prior treatment (e.g., laser photocoagulation, photodynamic therapy, intravitreal injections of steroids, or antivascular endothelial growth factor); and high myopia (>6 diopters). Our study was performed in agreement with French bioethical legislation and the Declaration of Helsinki for research involving human subjects. French Society of Ophthalmology
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Institutional Review Board approval was obtained for this study. All patients underwent a complete ophthalmologic examination that included best-corrected visual acuity using standard Early Treatment of Diabetic Retinopathy Study, fundus examination, cSLO fundus imaging (infrared [IR] reflectance or MultiColor [Heidelberg Engineering] mode), and tracked Spectralis SD-OCT (Spectralis real-time eye-tracking technology). Images prospectively collected were retrospectively evaluated with regard to cSLO fundus imaging and corresponding eye-tracked tomographic characteristics. The cSLO IR reflectance images are acquired with a 815-nm wavelength. MultiColor images are composed of 3 simultaneously acquired reflective images. Each one of these images is acquired with a distinctive cSLO wavelength: blue reflectance (486 nm), green reflectance (518 nm), and IR reflectance (815 nm). The final MultiColor image is an overlay of these 3 images. The IR and MultiColor images are acquired in real time, and the automatic real-time averaging can be used with both IR and MultiColor to minimize noise. The Spectralis high-resolution SDOCT system provides in vivo details of the anatomy of the retina that nearly resembles histologic specimens (optical axial image resolution of 7 mm). A new approach to improve depth imaging by OCT, termed “enhanced depth imaging” OCT, has been shown to reliably image the choroid.15 Enhanced depth imaging OCT uses a Spectralis SD-OCT positioned closer to the eye than usual such that a stable inverted image is produced. The net effect of this practice is that the sensitivity of the imaging in deeper layers of tissue is increased. The SD-OCT minimum acquisition protocol included 19 horizontal lines, each composed of 9 averaged OCT B-scans (1024 A-scans per line) at 240-mm intervals and covering a 66mm area; in a subset of eyes, further high-resolution 9-mm single B-scans (each composed of up to 100 averaged enhanced depth imaging OCT B-scans) were guided from the regressing drusen as visualized on IR and MultiColor images (yellow-gray deposits with interspersed refractile areas). The SD-OCT images consecutively collected were proportionally magnified for better visualization of sub-RPE changes and viewed with the contained Heidelberg Eye Explorer software (version 1.7.0.0, Heidelberg Engineering). Qualitative description was independently performed by 2 senior retinal physicians (readers: G.Q. and E.H.S.) on the highquality confidently tracked (IR and MultiColor images) SD-OCT scans. Disagreement between readers regarding the detection of features was resolved by open adjudication. To describe the SDOCT images, according to Spaide and Curcio,16 the following correspondence has been applied to the outer retinal layers: The innermost band reflects the external limiting membrane; a second band corresponds to the photoreceptors’ inner segment ellipsoid portion/outer segment (OS) interface, also known as the “ellipsoid zone”17; a third band represents the RPE/OS junction, also known as “interdigitation zone”17; and the most external band corresponds to the RPE/Bruch’s membrane complex. To interpret the multilaminar sub-RPE hyperreflectivity, according to Bloom and Singal,18 the hyperreflective band underlying areas of RPE elevation was defined as the outer Bruch’s membrane (oBM) layer. When available, previous SD-OCT examinations (matching areas of regressing drusen) were investigated in regard to sub-RPE reflectivity and time to development of multilaminar hyperreflectivity. Spectralis SD-OCT allows confidence in detecting and assessing small changes over time by using cSLO technology to track the eye and guide OCT to the selected location. By using a selected prior reference scan, the Spectralis SD-OCT aligns the reference fundus image with the live patient fundus image at
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Figure 1. Three different types of sub-retinal pigment epithelium (RPE) hyperreflectivity. A, Type 1 multilaminar hyperreflectivity (arrows) originating from what we interpreted as the inner Bruch’s membrane (iBM) layer. B, Type 2 multilaminar hyperreflectivity (arrows) originating from the outer Bruch’s membrane (oBM) layer. C, Type 3 multilaminar fragmented hyperreflectivity (arrows) originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation.
follow-up. The eye tracker recognizes the retina and then directs the SD-OCT scan to the same location. This eliminates the potential bias of subjective placement of the scan by the operator. The statistical analysis included descriptive statistics for demographic data and a qualitative description of the findings. Data were analyzed with the SPSS software version 20.0 for Mac (IBM/SPSS, Inc., Chicago, IL).
Results A total of 30 eyes of 23 consecutive patients (8 male and 15 female; mean age, 82.710.1 years; range, 71e98 years) showing on SD-OCT an intense multilaminar sub-RPE hyperreflectivity that matched with regressing calcific drusen as visualized by IR and MultiColor images (yellow-gray deposits with interspersed refractile areas) were included in this study. Seven patients had bilateral
lesions, and 15 of 30 eyes had multifocal lesions in the same eye. Best-corrected visual acuity ranged from 20/25 to 20/160. Overall, 142 of 626 SD-OCT scans showed at least 1 multilaminar sub-RPE hyperreflectivity and thus were selected for analysis. On SD-OCT, the multilaminar hyperreflectivity was found localize beneath the RPE and above the oBM layer but was never detected in the neurosensory retina, in the subretinal space, or beneath the Bruch’s membrane (in the choroid or sclera). A mean of 1.2 multilaminar sub-RPE hyperreflectivities per SD-OCT scan were identified by the 2 readers (k statistics ¼ 0.88; P < 0.001). The hyperreflective laminae varied from straight to slightly curvilinear. Despite the calcific appearance of regressing drusen on both fundus examination and IR/MultiColor images, not all the multilaminar hyperreflectivities produced a posterior optical shadowing of the underlying tissues. The SD-OCT analysis allowed the 2 readers to describe 3 different types of
Figure 2. Combined MultiColor (Heidelberg Engineering, Heidelberg, Germany) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT (right) shows an intense laminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
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Figure 3. Combined MultiColor image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT image (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
Figure 4. Combined infrared (IR) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
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sub-RPE hyperreflectivities (Fig 1). “Type 1” laminar/multilaminar hyper-reflectivity (Figs 2e4; Fig 5, available at http://aaojournal. org) was found in 24 scans of 12 eyes (k statistics ¼ 0.89; P < 0.001), characterized by an intense signal originating from what we interpreted as the inner Bruch’s membrane (iBM) layer. “Type 2” multilaminar hyperreflectivity (Fig 6; Figs 7e10, available at http://aaojournal.org) was found in 130 scans of 27 eyes (k statistics ¼ 0.91; P < 0.001), characterized by an intense signal originating from the oBM layer. “Type 3” multilaminar fragmented hyperreflectivity (Fig 10, available at http:// aaojournal.org, and Fig 11) was found in 22 scans of 11 eyes (k statistics ¼ 0.85; P < 0.01), characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. In 12 eyes of 12 patients for whom previous SD-OCT examinations (matching areas of regressing drusen) were available, multilaminar hyperreflectivities developed after a mean of 15.110.1 months (Figs 8 and 9, available at http://aaojournal.org).
Discussion In this study, we describe a multilaminar sub-RPE intense hyperreflectivity observed in vivo by SD-OCT in eyes clinically diagnosed with regressing drusen (documented by retrospective evaluation of progression) due to nonneovascular AMD. The SD-OCT analysis allowed us to identify 3 different types of sub-RPE hyperreflectivities: “type 1” laminar/multilaminar hyperreflectivity, characterized by an intense signal originating from what we interpreted as the iBM layer; “type 2” multilaminar hyperreflectivity, characterized by an intense signal originating from the oBM layer; and “type 3” multilaminar fragmented hyper-reflectivity, characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. In all scans (n ¼ 142) showing at least 1 of the 3 different types of intense multilaminar sub-RPE hyperreflectivity, the tracked SD-OCT matched with regressing calcific drusen as visualized by cSLO IR and MultiColor images (yellow-gray deposits with interspersed refractile areas). Because we have no clinicopathologic correlations for these findings, speculation about the actual localization and possible origin/composition is based on insights provided by multimodal imaging (combined cSLO IR/ MultiColor images and SD-OCT). Large drusen in eyes with AMD consist of sub-RPE mounds of cholesterol-enriched (w40% of drusen content) membranous debris (a lipid-rich material).9,10 Membranous debris is more highly unesterified cholesterol-enriched than expected if this material is composed of OS or RPE membranes only, suggesting not only a plasma source but also synthesis and assembly of an apoB-lipoprotein by RPE for cholesterol in the sub-RPE space.9 This mechanism is similar to what has been proposed for the origin of atherosclerotic liposomes: cholesterol excreted from intimal cells, apoB-lipoproteins of plasma origin modified in the extracellular space, or partly hydrolyzed chylomicron remnant particles.19e22 In drusen “maturation,” several layers of coiled membranous debris accumulate between the RPE and the Bruch’s membrane (internal and external to the basement membrane).5 Close to areas of atrophy, the mounds of membranous debris appear fused: The membranes pass
through the basement membrane to separate it from the inner collagenous layer.5 It is noteworthy that in drusen regression there is evidence not only of reduced formation of membranous debris but also of its removal by macrophage. Similar to what occurs in atherosclerosis (similar composition/progression and reflectivity),23 material not removed develops calcification.5 The stratified multilaminar sub-RPE intense hyperreflectivity in the current study described by SD-OCT in eyes clinically diagnosed with regressing drusen could be interpreted as the in vivo visualization of calcific coiled (not removed) membranous debris. Layers of lipid mineralization (in part similar to atherosclerotic changes) may develop internal and external to the basement membrane in the form of type 1 and type 2 multilaminar hyperreflectivity (originating from the iBM and oBM SD-OCT layers), respectively. Type 3 multilaminar fragmented hyperreflectivities (originating from both the iBM and the oBM SD-OCT layers) may correspond to fractures in the Bruch’s membrane, which develop after calcification and could lead to neovascularization.24,25 To the best of our knowledge, this is the first study to describe a stratified multilaminar sub-RPE intense hyperreflectivity in eyes with regressing drusen. Mukkamala et al26 described an OCT finding of layered hyperreflective bands beneath the RPE in eyes with vascularized pigment epithelial detachment that the authors called the “onion sign.” The onion sign was usually associated with chronic exudation from type 1 neovascularization in patients with AMD. Because the onion sign colocalized to areas of exudation that are known to consist of lipoprotein, the authors proposed that it may represent layers of precipitated lipid in the sub-RPE space; such precipitated (possibly with calcification) lipid also may be found in the neurosensory (intracystic) retina (Fig 12, available at http://aaojournal.org). In our study, multilaminar sub-RPE intense hyperreflectivity appeared fairly similar to the onion sign. This favors the idea that SD-OCT may allow visualization of precipitated lipid in the sub-RPE space, not only in neovascular AMD but also in dry AMD. In the current study, all types of SD-OCT multilaminar sub-RPE hyperreflectivities colocalized to IR/MultiColor refractile lesions (regressing drusen), which histologically are largely composed of lipid (i.e., membranous debris, also called lipoprotein-derived debris) developing calcification (precipitated lipid mineralization due to reduced removal by macrophage).5 Another possible explanation for the multilaminar subRPE hyperreflectivity in our series might be the presence of subclinical focal neovascularization (not distinguishable by fluorescein angiography and indocyanine green angiography) localized within regressing drusen. Our findings share similarities with the recently reported layers of tissue within vascularized pigment epithelium detachment (just beneath the RPE layer), which Spaide27 suggested to represent sub-RPE neovessels. This may also explain the iBM/oBM disjunction found in our series.
Study Limitation The main limitation of this study is the lack of corroboration by histologic studies or other in vivo high-resolution
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Figure 6. MultiColor image and combined infrared (IR) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 2 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and IR-tracked SD-OCT image (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the outer Bruch’s membrane (oBM) layer, and reaching the inner Bruch’s membrane (iBM).
imaging that confirms or contradicts these findings. Therefore, this interpretation needs to be confirmed by future research. In conclusion, we describe a novel SD-OCT finding appearing as multilaminar sub-RPE intense hyperreflectivity observed in vivo in eyes with regressing drusen. This multilaminar sub-RPE hyperreflectivity could be
interpreted as layers of lipid mineralization (membranous debris, also called lipoprotein-derived debris, developing calcification), internal and external to the basement membrane, with different degrees of fragmentation. To our knowledge, this is the first study of sub-RPE lipid mineralization in nonneovascular AMD that can be detected on SD-OCT examination.
Figure 11. Combined MultiColor image and spectral-domain optical coherence tomography (SD-OCT) scan of type 3 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT image (right) shows a multilaminar fragmented hyperreflectivity, apparently originating from both the inner Bruch’s membrane (iBM) and the outer Bruch’s membrane (oBM) layers.
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References 1. Spaide RF, Armstrong D, Browne R. Continuing medical education review: choroidal neovascularization in age-related macular degenerationewhat is the cause? Retina 2003;23:595–614. 2. Pauleikhoff D, Barondes MJ, Minassian D, et al. Drusen as risk factors in age-related macular disease. Am J Ophthalmol 1990;109:38–43. 3. Spaide RF, Curcio CA. Drusen characterization with multimodal imaging. Retina 2010;30:1441–54. 4. Hageman GS, Luthert PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res 2001;20:705–32. 5. Sarks JP, Sarks SH, Killingsworth MC. Evolution of geographic atrophy of the retinal pigment epithelium. Eye 1988;2:552–77. 6. Curcio CA, Medeiros NE, Millican CL. The Alabama AgeRelated Macular Degeneration Grading System for donor eyes. Invest Ophthalmol Vis Sci 1998;39:1085–96. 7. Green WR. Histopathology of age-related macular degeneration. Mol Vis 1999;5:27. 8. Marshall G, Konstas A, Reid G, et al. Collagens in the aged human macula. Graefes Arch Clin Exp Ophthalmol 1994;232: 133–40. 9. Curcio CA, Presley JB, Malek G, et al. Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy. Exp Eye Res 2005;81:731–41. 10. Wang L, Clark ME, Crossman DK, et al. Abundant lipid and protein components of drusen. PLoS ONE 2010;5:e10329. 11. Curcio CA, Millican CL. Basal linear deposit and large drusen are specific for early age-related maculopathy. Arch Ophthalmol 1999;117:329–39. 12. Sarks S, Cherepanoff S, Killingsworth M, Sarks J. Relationship of basal laminar deposit and membranous debris to the clinical presentation of early age-related macular degeneration. Invest Ophthalmol Vis Sci 2007;48:968–77. 13. Curcio CA, Johnson M, Huang J-D, Rudolf M. Aging, agerelated macular degeneration, and the response-to-retention of apolipoprotein B-containing lipoproteins. Prog Ret Eye Res 2009;28:393–422. 14. Klein R, Klein BE, Knudtson MD, et al. Fifteen-year cumulative incidence of age-related macular degeneration: the Beaver Dam Eye Study. Ophthalmology 2007;114:253–62.
15. Spaide RF, Koizumi H, Pozonni MC. Enhanced depth imaging spectral-domain optical coherence tomography. Am J Ophthalmol 2008;146:496–500. 16. Spaide RF, Curcio CA. Anatomical correlates to the bands seen in the outer retina by optical coherence tomography: literature review and model. Retina 2011;31:1609–19. 17. Spaide RF. Questioning optical coherence tomography. Ophthalmology 2012;119:2203–04.e1. 18. Bloom SM, Singal IP. The outer Bruch membrane layer: a previously undescribed spectral-domain optical coherence tomography finding. Retina 2011;31:316–23. 19. Chao FF, Blanchette-Mackie E, Chen Y-J, et al. Characterization of two unique cholesterol-rich lipid particles isolated from human atherosclerotic lesions. Am J Pathol 1990;136:169–79. 20. Chung BH, Tallis G, Yalamoori V, et al. Liposome-like particles isolated from human atherosclerotic plaques are structurally and compositionally similar to surface remnants of triglyceride-rich lipoproteins. Arterioscler Thromb 1994;14: 622–35. 21. Kruth HS, Ifrim I, Chang J, et al. Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking. J Lipid Res 2001;42: 1492–500. 22. Robenek H, Schmitz G. Caþþ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. II. Characterization of intracellular morphologic changes. Arteriosclerosis 1988;8:57–67. 23. Nair A, Kuban BD, Tuzcu EM, et al. Coronary plaque classification with intravascular ultrasound radiofrequency data analysis. Circulation 2002;106:2200–6. 24. Spraul CW, Lang GE, Grossniklaus HE, Lang GK. Histologic and morphometric analysis of the choroid, Bruch’s membrane, and retinal pigment epithelium in postmortem eyes with agerelated macular degeneration and histologic examination of surgically excised choroidal neovascular membranes. Surv Ophthalmol 1999;44(Suppl 1):S10–32. 25. Miller JW. Age-related macular degeneration revisitede piecing the puzzle: the LXIX Edward Jackson memorial lecture. Am J Ophthalmol 2013;155:1–35. 26. Mukkamala SK, Costa RA, Fung A, et al. Optical coherence tomographic imaging of sub-retinal pigment epithelium lipid. Arch Ophthalmol 2012;130:1547–53. 27. Spaide RF. Enhanced depth imaging optical coherence tomography of retinal pigment epithelial detachment in agerelated macular degeneration. Am J Ophthalmol 2009;147: 644–52.
Footnotes and Financial Disclosures Originally received: March 4, 2013. Final revision: June 12, 2013. Accepted: June 12, 2013. Available online: July 26, 2013.
Manuscript no. 2013-353.
Correspondence: Giuseppe Querques, MD, PhD, Department of Ophthalmology, Centre Hospitalier Intercommunal de Creteil, 40 Avenue de Verdun, 94000 Creteil, France. E-mail: [email protected].
Department of Ophthalmology, Centre Hospitalier Intercommunal de Creteil University Paris Est Creteil, Creteil, France. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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Deposition of extracellular material between the basal lamina of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch’s membrane is generally referred to as “drusen.”1 Drusen usually appear after the age of 50 years and are considered to be the main risk factors of age-related macular degeneration (AMD).2 Large AMD drusen are yellow-white, mound-like RPE elevations typically 63 to 1000 mm in diameter.3 Histopathologic investigations showed that large drusen are mounds external to the RPE basement membrane containing membranous debris (also called “lipoproteinderived debris”), a neutral lipid-rich material.3 Basal linear deposits and large drusen should be regarded as the same lesion.4e6 A basal linear deposit consists of a thin, relatively extensive layer of membranous profiles.7 This is in contradistinction to a basal laminar deposit, a diffusely 2014 by the American Academy of Ophthalmology Published by Elsevier Inc.
deposited material that lies internal to the RPE basement membrane, and is composed of fibrils, amorphous material, and a banded component that is reminiscent of long-spacing collagen.8 Drusen growth is characterized by the continued deposition (overproduction/underclearance) of drusen-associated constituents (membranous debris). Early complexes within Bruch’s membrane, such as immune complexes, or other local ligands might serve as “nucleation sites” for the deposition of additional self-aggregating proteins or lipids.4 Curcio et al9 demonstrated that Folch-extractable lipid, dominated by esterified and unesterified cholesterol, enriches drusen and basal deposits in human eyes with AMD (w40% of drusen content).10 A source for cholesterol in the sub-RPE space may be plasma, endogenous synthesis, ISSN 0161-6420/14/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.06.024
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photoreceptor disk shedding, and assembly of an apolipoprotein B (apoB)-lipoprotein by RPE. In drusen “maturation,” several layers of coiled membranes lay between the RPE and its basement membrane (membranous debris crossing the basal laminar deposit in strands or mounds).5,11,12 These membranous debris extend inward between adjacent RPE cells. A thin stratus of membranes also lay external to the basement membrane. Regressing drusen show evidence of not only reduced formation of membranous debris but also its elimination by macrophages. Material that is not removed develops calcification.5 A better understanding of the physiopathologic features of drusen is required to develop rational and effective AMD therapies. Drusen size, type, and composition reveal both past formative processes4,13,14 and future progression of disease.4,13,14 Because of the paucity of histopathologic information about eyes previously imaged in the clinic, we could refer to contemporary multimodal, high-resolution in vivo imaging to interpret differences in drusen appearance. Spectralis high-resolution spectral-domain (SD) optical coherence tomography (OCT) (Spectralis SD-OCT; Heidelberg Engineering, Heidelberg, Germany) is a high-speed OCT system (up to 40 000 axial scans per second) using SD/ Fourier domain detection, with an optical axial image resolution of 7 mm (digital axial image resolution of 3.5 mm). In Spectralis SD-OCT, confocal scanning laser ophthalmoscopy (cSLO) with real-time eye tracking technology allows one to track the eye and guide OCT to the selected location, thus giving a real-time reference for locating the SD-OCT scan. The purpose of this study was to describe and interpret a multilaminar sub-RPE intense hyperreflectivity observed in vivo in eyes clinically diagnosed with regressing drusen due to nonneovascular AMD.
Methods Twenty-three patients clinically diagnosed with regressing drusen (documented by retrospective evaluation of progression) due to nonneovascular AMD who consecutively presented at the Retinal Service of the University Eye Clinic of Creteil between September and December 2012 were submitted to cSLO fundus imaging and “eye-tracked” SD-OCT. Inclusion criteria were age >50 years; presence on fundus biomicroscopy of soft indistinct drusen combined with RPE changes and regressing calcific drusen (appearing as refractile lesions) in the macular area as manifestation of nonexudative AMD, with no evidence of choroidal neovascularization on fluorescein angiography and indocyanine green angiography; no intraretinal or subretinal fluid; and no sub-RPE/ retinal hemorrhage. Exclusion criteria were signs of any other retinal disease in the study eye, such as retinal vascular disease (i.e., diabetic retinopathy and retinal vein occlusion) or vitreoretinal disease (i.e., vitreomacular traction syndrome and epiretinal membrane; presence of choroidal neovascularization (i.e., exudative AMD; any prior treatment (e.g., laser photocoagulation, photodynamic therapy, intravitreal injections of steroids, or antivascular endothelial growth factor); and high myopia (>6 diopters). Our study was performed in agreement with French bioethical legislation and the Declaration of Helsinki for research involving human subjects. French Society of Ophthalmology
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Institutional Review Board approval was obtained for this study. All patients underwent a complete ophthalmologic examination that included best-corrected visual acuity using standard Early Treatment of Diabetic Retinopathy Study, fundus examination, cSLO fundus imaging (infrared [IR] reflectance or MultiColor [Heidelberg Engineering] mode), and tracked Spectralis SD-OCT (Spectralis real-time eye-tracking technology). Images prospectively collected were retrospectively evaluated with regard to cSLO fundus imaging and corresponding eye-tracked tomographic characteristics. The cSLO IR reflectance images are acquired with a 815-nm wavelength. MultiColor images are composed of 3 simultaneously acquired reflective images. Each one of these images is acquired with a distinctive cSLO wavelength: blue reflectance (486 nm), green reflectance (518 nm), and IR reflectance (815 nm). The final MultiColor image is an overlay of these 3 images. The IR and MultiColor images are acquired in real time, and the automatic real-time averaging can be used with both IR and MultiColor to minimize noise. The Spectralis high-resolution SDOCT system provides in vivo details of the anatomy of the retina that nearly resembles histologic specimens (optical axial image resolution of 7 mm). A new approach to improve depth imaging by OCT, termed “enhanced depth imaging” OCT, has been shown to reliably image the choroid.15 Enhanced depth imaging OCT uses a Spectralis SD-OCT positioned closer to the eye than usual such that a stable inverted image is produced. The net effect of this practice is that the sensitivity of the imaging in deeper layers of tissue is increased. The SD-OCT minimum acquisition protocol included 19 horizontal lines, each composed of 9 averaged OCT B-scans (1024 A-scans per line) at 240-mm intervals and covering a 66mm area; in a subset of eyes, further high-resolution 9-mm single B-scans (each composed of up to 100 averaged enhanced depth imaging OCT B-scans) were guided from the regressing drusen as visualized on IR and MultiColor images (yellow-gray deposits with interspersed refractile areas). The SD-OCT images consecutively collected were proportionally magnified for better visualization of sub-RPE changes and viewed with the contained Heidelberg Eye Explorer software (version 1.7.0.0, Heidelberg Engineering). Qualitative description was independently performed by 2 senior retinal physicians (readers: G.Q. and E.H.S.) on the highquality confidently tracked (IR and MultiColor images) SD-OCT scans. Disagreement between readers regarding the detection of features was resolved by open adjudication. To describe the SDOCT images, according to Spaide and Curcio,16 the following correspondence has been applied to the outer retinal layers: The innermost band reflects the external limiting membrane; a second band corresponds to the photoreceptors’ inner segment ellipsoid portion/outer segment (OS) interface, also known as the “ellipsoid zone”17; a third band represents the RPE/OS junction, also known as “interdigitation zone”17; and the most external band corresponds to the RPE/Bruch’s membrane complex. To interpret the multilaminar sub-RPE hyperreflectivity, according to Bloom and Singal,18 the hyperreflective band underlying areas of RPE elevation was defined as the outer Bruch’s membrane (oBM) layer. When available, previous SD-OCT examinations (matching areas of regressing drusen) were investigated in regard to sub-RPE reflectivity and time to development of multilaminar hyperreflectivity. Spectralis SD-OCT allows confidence in detecting and assessing small changes over time by using cSLO technology to track the eye and guide OCT to the selected location. By using a selected prior reference scan, the Spectralis SD-OCT aligns the reference fundus image with the live patient fundus image at
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Figure 1. Three different types of sub-retinal pigment epithelium (RPE) hyperreflectivity. A, Type 1 multilaminar hyperreflectivity (arrows) originating from what we interpreted as the inner Bruch’s membrane (iBM) layer. B, Type 2 multilaminar hyperreflectivity (arrows) originating from the outer Bruch’s membrane (oBM) layer. C, Type 3 multilaminar fragmented hyperreflectivity (arrows) originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation.
follow-up. The eye tracker recognizes the retina and then directs the SD-OCT scan to the same location. This eliminates the potential bias of subjective placement of the scan by the operator. The statistical analysis included descriptive statistics for demographic data and a qualitative description of the findings. Data were analyzed with the SPSS software version 20.0 for Mac (IBM/SPSS, Inc., Chicago, IL).
Results A total of 30 eyes of 23 consecutive patients (8 male and 15 female; mean age, 82.710.1 years; range, 71e98 years) showing on SD-OCT an intense multilaminar sub-RPE hyperreflectivity that matched with regressing calcific drusen as visualized by IR and MultiColor images (yellow-gray deposits with interspersed refractile areas) were included in this study. Seven patients had bilateral
lesions, and 15 of 30 eyes had multifocal lesions in the same eye. Best-corrected visual acuity ranged from 20/25 to 20/160. Overall, 142 of 626 SD-OCT scans showed at least 1 multilaminar sub-RPE hyperreflectivity and thus were selected for analysis. On SD-OCT, the multilaminar hyperreflectivity was found localize beneath the RPE and above the oBM layer but was never detected in the neurosensory retina, in the subretinal space, or beneath the Bruch’s membrane (in the choroid or sclera). A mean of 1.2 multilaminar sub-RPE hyperreflectivities per SD-OCT scan were identified by the 2 readers (k statistics ¼ 0.88; P < 0.001). The hyperreflective laminae varied from straight to slightly curvilinear. Despite the calcific appearance of regressing drusen on both fundus examination and IR/MultiColor images, not all the multilaminar hyperreflectivities produced a posterior optical shadowing of the underlying tissues. The SD-OCT analysis allowed the 2 readers to describe 3 different types of
Figure 2. Combined MultiColor (Heidelberg Engineering, Heidelberg, Germany) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT (right) shows an intense laminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
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Figure 3. Combined MultiColor image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT image (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
Figure 4. Combined infrared (IR) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 1 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the inner Bruch’s membrane (iBM) layer (note the underlying outer Bruch’s membrane [oBM] layer).
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sub-RPE hyperreflectivities (Fig 1). “Type 1” laminar/multilaminar hyper-reflectivity (Figs 2e4; Fig 5, available at http://aaojournal. org) was found in 24 scans of 12 eyes (k statistics ¼ 0.89; P < 0.001), characterized by an intense signal originating from what we interpreted as the inner Bruch’s membrane (iBM) layer. “Type 2” multilaminar hyperreflectivity (Fig 6; Figs 7e10, available at http://aaojournal.org) was found in 130 scans of 27 eyes (k statistics ¼ 0.91; P < 0.001), characterized by an intense signal originating from the oBM layer. “Type 3” multilaminar fragmented hyperreflectivity (Fig 10, available at http:// aaojournal.org, and Fig 11) was found in 22 scans of 11 eyes (k statistics ¼ 0.85; P < 0.01), characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. In 12 eyes of 12 patients for whom previous SD-OCT examinations (matching areas of regressing drusen) were available, multilaminar hyperreflectivities developed after a mean of 15.110.1 months (Figs 8 and 9, available at http://aaojournal.org).
Discussion In this study, we describe a multilaminar sub-RPE intense hyperreflectivity observed in vivo by SD-OCT in eyes clinically diagnosed with regressing drusen (documented by retrospective evaluation of progression) due to nonneovascular AMD. The SD-OCT analysis allowed us to identify 3 different types of sub-RPE hyperreflectivities: “type 1” laminar/multilaminar hyperreflectivity, characterized by an intense signal originating from what we interpreted as the iBM layer; “type 2” multilaminar hyperreflectivity, characterized by an intense signal originating from the oBM layer; and “type 3” multilaminar fragmented hyper-reflectivity, characterized by an intense signal originating from what we interpreted as both the iBM and the oBM, showing different degrees of fragmentation. In all scans (n ¼ 142) showing at least 1 of the 3 different types of intense multilaminar sub-RPE hyperreflectivity, the tracked SD-OCT matched with regressing calcific drusen as visualized by cSLO IR and MultiColor images (yellow-gray deposits with interspersed refractile areas). Because we have no clinicopathologic correlations for these findings, speculation about the actual localization and possible origin/composition is based on insights provided by multimodal imaging (combined cSLO IR/ MultiColor images and SD-OCT). Large drusen in eyes with AMD consist of sub-RPE mounds of cholesterol-enriched (w40% of drusen content) membranous debris (a lipid-rich material).9,10 Membranous debris is more highly unesterified cholesterol-enriched than expected if this material is composed of OS or RPE membranes only, suggesting not only a plasma source but also synthesis and assembly of an apoB-lipoprotein by RPE for cholesterol in the sub-RPE space.9 This mechanism is similar to what has been proposed for the origin of atherosclerotic liposomes: cholesterol excreted from intimal cells, apoB-lipoproteins of plasma origin modified in the extracellular space, or partly hydrolyzed chylomicron remnant particles.19e22 In drusen “maturation,” several layers of coiled membranous debris accumulate between the RPE and the Bruch’s membrane (internal and external to the basement membrane).5 Close to areas of atrophy, the mounds of membranous debris appear fused: The membranes pass
through the basement membrane to separate it from the inner collagenous layer.5 It is noteworthy that in drusen regression there is evidence not only of reduced formation of membranous debris but also of its removal by macrophage. Similar to what occurs in atherosclerosis (similar composition/progression and reflectivity),23 material not removed develops calcification.5 The stratified multilaminar sub-RPE intense hyperreflectivity in the current study described by SD-OCT in eyes clinically diagnosed with regressing drusen could be interpreted as the in vivo visualization of calcific coiled (not removed) membranous debris. Layers of lipid mineralization (in part similar to atherosclerotic changes) may develop internal and external to the basement membrane in the form of type 1 and type 2 multilaminar hyperreflectivity (originating from the iBM and oBM SD-OCT layers), respectively. Type 3 multilaminar fragmented hyperreflectivities (originating from both the iBM and the oBM SD-OCT layers) may correspond to fractures in the Bruch’s membrane, which develop after calcification and could lead to neovascularization.24,25 To the best of our knowledge, this is the first study to describe a stratified multilaminar sub-RPE intense hyperreflectivity in eyes with regressing drusen. Mukkamala et al26 described an OCT finding of layered hyperreflective bands beneath the RPE in eyes with vascularized pigment epithelial detachment that the authors called the “onion sign.” The onion sign was usually associated with chronic exudation from type 1 neovascularization in patients with AMD. Because the onion sign colocalized to areas of exudation that are known to consist of lipoprotein, the authors proposed that it may represent layers of precipitated lipid in the sub-RPE space; such precipitated (possibly with calcification) lipid also may be found in the neurosensory (intracystic) retina (Fig 12, available at http://aaojournal.org). In our study, multilaminar sub-RPE intense hyperreflectivity appeared fairly similar to the onion sign. This favors the idea that SD-OCT may allow visualization of precipitated lipid in the sub-RPE space, not only in neovascular AMD but also in dry AMD. In the current study, all types of SD-OCT multilaminar sub-RPE hyperreflectivities colocalized to IR/MultiColor refractile lesions (regressing drusen), which histologically are largely composed of lipid (i.e., membranous debris, also called lipoprotein-derived debris) developing calcification (precipitated lipid mineralization due to reduced removal by macrophage).5 Another possible explanation for the multilaminar subRPE hyperreflectivity in our series might be the presence of subclinical focal neovascularization (not distinguishable by fluorescein angiography and indocyanine green angiography) localized within regressing drusen. Our findings share similarities with the recently reported layers of tissue within vascularized pigment epithelium detachment (just beneath the RPE layer), which Spaide27 suggested to represent sub-RPE neovessels. This may also explain the iBM/oBM disjunction found in our series.
Study Limitation The main limitation of this study is the lack of corroboration by histologic studies or other in vivo high-resolution
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Figure 6. MultiColor image and combined infrared (IR) image and spectral-domain optical coherence tomography (SD-OCT) scan of type 2 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and IR-tracked SD-OCT image (right) shows an intense multilaminar hyperreflective signal (arrows) beneath the retinal pigment epithelium (RPE) layer, apparently originating from the outer Bruch’s membrane (oBM) layer, and reaching the inner Bruch’s membrane (iBM).
imaging that confirms or contradicts these findings. Therefore, this interpretation needs to be confirmed by future research. In conclusion, we describe a novel SD-OCT finding appearing as multilaminar sub-RPE intense hyperreflectivity observed in vivo in eyes with regressing drusen. This multilaminar sub-RPE hyperreflectivity could be
interpreted as layers of lipid mineralization (membranous debris, also called lipoprotein-derived debris, developing calcification), internal and external to the basement membrane, with different degrees of fragmentation. To our knowledge, this is the first study of sub-RPE lipid mineralization in nonneovascular AMD that can be detected on SD-OCT examination.
Figure 11. Combined MultiColor image and spectral-domain optical coherence tomography (SD-OCT) scan of type 3 multilaminar hyperreflectivity in a patient with nonexudative age-related macular degeneration. MultiColor image (left) shows a regressing calcific drusen (arrowhead), and tracked SD-OCT image (right) shows a multilaminar fragmented hyperreflectivity, apparently originating from both the inner Bruch’s membrane (iBM) and the outer Bruch’s membrane (oBM) layers.
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15. Spaide RF, Koizumi H, Pozonni MC. Enhanced depth imaging spectral-domain optical coherence tomography. Am J Ophthalmol 2008;146:496–500. 16. Spaide RF, Curcio CA. Anatomical correlates to the bands seen in the outer retina by optical coherence tomography: literature review and model. Retina 2011;31:1609–19. 17. Spaide RF. Questioning optical coherence tomography. Ophthalmology 2012;119:2203–04.e1. 18. Bloom SM, Singal IP. The outer Bruch membrane layer: a previously undescribed spectral-domain optical coherence tomography finding. Retina 2011;31:316–23. 19. Chao FF, Blanchette-Mackie E, Chen Y-J, et al. Characterization of two unique cholesterol-rich lipid particles isolated from human atherosclerotic lesions. Am J Pathol 1990;136:169–79. 20. Chung BH, Tallis G, Yalamoori V, et al. Liposome-like particles isolated from human atherosclerotic plaques are structurally and compositionally similar to surface remnants of triglyceride-rich lipoproteins. Arterioscler Thromb 1994;14: 622–35. 21. Kruth HS, Ifrim I, Chang J, et al. Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking. J Lipid Res 2001;42: 1492–500. 22. Robenek H, Schmitz G. Caþþ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. II. Characterization of intracellular morphologic changes. Arteriosclerosis 1988;8:57–67. 23. Nair A, Kuban BD, Tuzcu EM, et al. Coronary plaque classification with intravascular ultrasound radiofrequency data analysis. Circulation 2002;106:2200–6. 24. Spraul CW, Lang GE, Grossniklaus HE, Lang GK. Histologic and morphometric analysis of the choroid, Bruch’s membrane, and retinal pigment epithelium in postmortem eyes with agerelated macular degeneration and histologic examination of surgically excised choroidal neovascular membranes. Surv Ophthalmol 1999;44(Suppl 1):S10–32. 25. Miller JW. Age-related macular degeneration revisitede piecing the puzzle: the LXIX Edward Jackson memorial lecture. Am J Ophthalmol 2013;155:1–35. 26. Mukkamala SK, Costa RA, Fung A, et al. Optical coherence tomographic imaging of sub-retinal pigment epithelium lipid. Arch Ophthalmol 2012;130:1547–53. 27. Spaide RF. Enhanced depth imaging optical coherence tomography of retinal pigment epithelial detachment in agerelated macular degeneration. Am J Ophthalmol 2009;147: 644–52.
Footnotes and Financial Disclosures Originally received: March 4, 2013. Final revision: June 12, 2013. Accepted: June 12, 2013. Available online: July 26, 2013.
Manuscript no. 2013-353.
Correspondence: Giuseppe Querques, MD, PhD, Department of Ophthalmology, Centre Hospitalier Intercommunal de Creteil, 40 Avenue de Verdun, 94000 Creteil, France. E-mail: [email protected].
Department of Ophthalmology, Centre Hospitalier Intercommunal de Creteil University Paris Est Creteil, Creteil, France. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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