Quantitative Autofluorescence at AMD's Beginnings Highlights Retinal Topography and Grading System Differences: ALSTAR2 Baseline.

INTRODUCTION: The aims of the study were to describe baseline quantitative (short-wavelength) autofluorescence (qAF) findings in a large pseudophakic cohort at age-related macular degeneration (AMD)'s beginnings and to assess qAF8 as an outcome measure and evaluate Age-Related Eye Disease Study (AREDS) and Beckman grading systems. METHODS: In the ALSTAR2 baseline cohort (NCT04112667), 346 pseudophakic eyes of 188 persons (74.0 ± 5.5 years) were classified as normal (N = 160 by AREDS, 158 by Beckman), early AMD (eAMD) (N = 104, 66), and intermediate AMD (iAMD) (N = 82, 122). Groups were compared via mean qAF intensities in a 6°-8° annulus (qAF8) and maps of differences between observations and the overall mean, divided by standard deviation (Z-score). RESULTS: qAF8 did not differ significantly among diagnostic groups by either stratification (p = 0.0869 AREDS; p = 0.0569 by Beckman). Notably, 45 eyes considered eAMD by AREDS became iAMD by Beckman. For AREDS-stratified eyes, Z-score maps showed higher centrally located qAF for normal, near the mean in eAMD, and lower values for iAMD. Maps deviated from this pattern for Beckman-stratified eyes. CONCLUSIONS: In a large sample of pseudophakic eyes, qAF8 does not differ overall from normal aging to iAMD but also does not capture the earliest AMD activity in the macula lutea. AREDS classification gives results more consistent with a slow decline in histologic autofluorescence than Beckman classification.

Imaging Histology Correlations of Intraretinal Fluid in Neovascular Age-Related Macular Degeneration.

Purpose: Fluid presence and dynamism is central to the diagnosis and management of neovascular age-related macular degeneration. On optical coherence tomography (OCT), some hyporeflective spaces arise through vascular permeability (exudation) and others arise through degeneration (transudation). Herein we determined whether the histological appearance of fluid manifested this heterogeneity. Methods: Two eyes of a White woman in her 90s with anti-vascular endothelial growth factor treated bilateral type 3 neovascularization secondary to age-related macular degeneration were osmicated, prepared for submicrometer epoxy resin sections, and correlated to eye-tracked spectral domain OCT. Examples of intraretinal tissue fluid were sought among similarly prepared donor eyes with fibrovascular scars, in a web-based age-related macular degeneration histopathology resource. Fluid stain intensity was quantified in reference to Bruch's membrane and the empty glass slide. Results: Exudative fluid by OCT was slightly reflective and dynamically responded to anti-vascular endothelial growth factor. On histology, this fluid stained moderately, possessed a smooth and homogenous texture, and contained blood cells and fibrin. Nonexudative fluid in degenerative cysts and in outer retinal tubulation was minimally reflective on OCT and did not respond to anti-vascular endothelial growth factor. By histology, this fluid stained lightly, possessed a finely granular texture, and contained mainly tissue debris. Quantification supported the qualitative impressions of fluid stain density. Cells containing retinal pigment epithelium organelles localized to both fluid types. Conclusions: High-resolution histology of osmicated tissue can distinguish between exudative and nonexudative fluid, some of which is transudative. Translational Relevance: OCT and histological features of different fluid types can inform clinical decision-making and assist in the interpretation of newly available automated fluid detection algorithms.

INFLAMMATORY CELL ACTIVITY IN TREATED NEOVASCULAR AGE-RELATED MACULAR DEGENERATION: A Histologic Case Study.

BACKGROUND: Imaging indicators of macular neovascularization risk can help determine patient eligibility for new treatments for geographic atrophy secondary to age-related macular degeneration. Because type 1 macular neovascularization includes inflammation, we assessed by histology the distribution of cells with inflammatory potential in two fellow eyes with age-related macular degeneration. METHODS: Two eyes of a White woman in her 90's with type 3 macular neovascularization treated with antivascular endothelial growth factor were prepared for high-resolution histology. Eye-tracked spectral domain optical coherence tomography applied to the preserved donor eyes linked in vivo imaging to histology. Cells were enumerated in the intraretinal, subretinal, and subretinal retinal pigment epithelium (RPE)-basal lamina compartments on 199 glass slides. Cells with numerous organelles were considered to RPE-derived; cells with sparse RPE organelles were considered non-RPE phagocytes. RESULTS: Both eyes had soft drusen and abundant subretinal drusenoid deposit. In the retina and subretinal space, RPE-derived cells, including hyperreflective foci, were common (n = 125 and 73, respectively). Non-RPE phagocytes were infrequent (n = 5 in both). Over drusen, RPE morphology transitioned smoothly from the age-normal layer toward the top, suggesting transdifferentiation. The sub-RPE-basal lamina space had RPE-derived cells (n = 87) and non-RPE phagocytes (n = 49), including macrophages and giant cells. CONCLUSION: Numerous sub-RPE-basal lamina cells of several types are consistent with the documented presence of proinflammatory lipids in drusen and aged Bruch's membrane. The relatively compartmentalized abundance of infiltrating cells suggests that drusen contents are more inflammatory than subretinal drusenoid deposit, perhaps reflecting their environments. Ectopic RPE occurs frequently. Some manifest as hyperreflective foci. More cells may be visible as optical coherence tomography technologies evolve.

Discernibility of the Interdigitation Zone (IZ), a Potential Optical Coherence Tomography (OCT) Biomarker for Visual Dysfunction in Aging.

PURPOSE: Photoreceptor (PR) outer segments, retinal pigment epithelium apical processes, and inter-PR matrix contribute to the interdigitation zone (IZ) of optical coherence tomography (OCT). We hypothesize that this interface degrades over adulthood, in concert with a delay of rod mediated dark adaptation (RMDA). To explore this idea, we determined IZ discernibility and RMDA in younger and older adults. METHODS: For this cross-sectional study, eyes of 20 young (20-30 years) and 40 older (≥60 years) participants with normal maculas according to the AREDS 9-step grading system underwent OCT imaging and RMDA testing at 5° superior to the fovea. Custom FIJI plugins enabled analysis for IZ discernibility at 9 eccentricities in 0.5 mm steps on one single horizontal B-scan through the fovea. Locations with discernible IZ met two criteria: visibility on B-scans and a distinct peak on a longitudinal reflectivity profile. The frequency of sites meeting both criteria was compared between both age groups and correlated with rod intercept time (RIT). RESULTS: The median number of locations with discernible IZ was significantly higher (foveal, 4 vs. 0, p = 0.0099; extra-foveal 6 vs. 0, p < 0.001) in eyes of young (26 ± 3 years) compared to older (73 ± 5 years) participants. For the combined young and older sample, the higher frequency of discernible IZ was correlated with shorter RIT (faster dark adaptation) (rs = -0.56, p < 0.0001). This association was significant within young eyes (rs = -0.54; p = 0.0134) and not within older eyes (rs = -0.29, p = 0.706). CONCLUSIONS: Results suggest that the interface between outer segments and apical processes degrades in normal aging, potentially contributing to delayed rod-mediated dark adaptation. More research is needed to verify an age-related association between IZ discernibility and rod-mediated dark adaptation. If confirmed in a large sample, IZ discernibility might prove to be a valuable biomarker and predictor for visual function in aging.

Quantitative Fundus Autofluorescence in Systemic Chloroquine/Hydroxychloroquine Therapy: One Year Follow-Up.

Purpose: Systemic chloroquine/hydroxychloroquine (CQ/HCQ) can cause severe ocular side effects including bull's eye maculopathy (BEM). Recently, we reported higher quantitative autofluorescence (QAF) levels in patients with CQ/HCQ intake. Here, QAF in patients taking CQ/HCQ in a 1-year follow-up is reported. Methods: Fifty-eight patients currently or previously treated with CQ/HCQ (cumulative doses 94-2435 g) and 32 age- and sex-matched healthy subjects underwent multimodal retinal imaging (infrared, red free, fundus autofluorescence [FAF], QAF [488 nm], and spectral-domain optical coherence tomography (SD-OCT). For analysis, custom written FIJI plugins were used for image processing, multimodal image stacks assembling, and QAF calculation. Results: Thirty patients (28 without BEM and 2 with BEM, age range = 25-69 years) were followed up (370 ± 63 days). QAF values in patients taking CQ/HCQ showed a significant increase between baseline and follow-up examination: 282.0 ± 67.9 to 297.7 ± 70.0 (QAF a.u.), P = 0.002. An increase up to 10% was observed in the superior macular hemisphere. Eight individuals (including 1 patient with BEM) had a pronounced QAF increase of up to 25%. Compared to healthy controls, QAF levels in patients taking CQ/HCQ were significantly increased (P = 0.04). Conclusions: Our study confirms our previous finding of increased QAF in patients taking CQ/HCQ with a further significant QAF increase from baseline to follow-up. Whether pronounced QAF increase might predispose for rapid progression toward structural changes and BEM development is currently investigated in ongoing studies. Translational Relevance: In addition to standard screening tools during systemic CQ/HCQ treatment, QAF imaging might be useful in CQ/HCQ monitoring and could serve as a screening tool in the future.

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged ≥80 years old, with a death-to-preservation time (DtoP) of ≤6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30° macula cube, 30 µm spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 ± 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Macular and Plasma Xanthophylls Are Higher in Age-related Macular Degeneration than in Normal Aging: Alabama Study on Early Age-related Macular Degeneration 2 Baseline.

Purpose: Quantification of retinal xanthophyll carotenoids in eyes with and without age-related macular degeneration (AMD) via macular pigment optical volume (MPOV), a metric for xanthophyll abundance from dual wavelength autofluorescence, plus correlations to plasma levels, could clarify the role of lutein (L) and zeaxanthin (Z) in health, AMD progression, and supplementation strategies. Design: Cross-sectional observational study (NCT04112667). Participants: Adults ≥ 60 years from a comprehensive ophthalmology clinic, with healthy maculas or maculas meeting fundus criteria for early or intermediate AMD. Methods: Macular health and supplement use was assessed by the Age-related Eye Disease Study (AREDS) 9-step scale and self-report, respectively. Macular pigment optical volume was measured from dual wavelength autofluorescence emissions (Spectralis, Heidelberg Engineering). Non-fasting blood draws were assayed for L and Z using high-performance liquid chromatography. Associations among plasma xanthophylls and MPOV were assessed adjusting for age. Main Outcome Measures: Age-related macular degeneration presence and severity, MPOV in fovea-centered regions of radius 2.0° and 9.0°; plasma L and Z (µM/ml). Results: Of 809 eyes from 434 persons (89% aged 60-79, 61% female), 53.3% eyes were normal, 28.2% early AMD, and 18.5% intermediate AMD. Macular pigment optical volume 2° and 9° were similar in phakic and pseudophakic eyes, which were combined for analysis. Macular pigment optical volume 2° and 9° and plasma L and Z were higher in early AMD than normal and higher still in intermediate AMD (P < 0.0001). For all participants, higher plasma L was correlated with higher MPOV 2° (Spearman correlation coefficient [Rs] = 0.49; P < 0.0001). These correlations were significant (P < 0.0001) but lower in normal (Rs = 0.37) than early and intermediate AMD (Rs = 0.52 and 0.51, respectively). Results were similar for MPOV 9°. Plasma Z, MPOV 2°, and MPOV 9° followed this same pattern of associations. Associations were not affected by supplement use or smoking status. Conclusions: A moderate positive correlation of MPOV with plasma L and Z comports with regulated xanthophyll bioavailability and a hypothesized role for xanthophyll transfer in soft drusen biology. An assumption that xanthophylls are low in AMD retina underlies supplementation strategies to reduce progression risk, which our data do not support. Whether higher xanthophyll levels in AMD are due to supplement use cannot be determined in this study.

OPTICAL COHERENCE TOMOGRAPHY FEATURES RELEVANT TO NEOVASCULAR AGE-RELATED MACULAR DEGENERATION MANAGEMENT AND NONNEOVASCULAR AGE-RELATED MACULAR DEGENERATION PROGRESSION: CLINICOPATHOLOGIC CORRELATION.

PURPOSE: Clinicopathologic correlation of two optical coherence tomography (OCT) features in neovascular age-related macular degeneration. METHODS: Case report, clinicopathologic correlation. RESULTS: A patient in her 90s was diagnosed with Type 3 macular neovascularization secondary to age-related macular degeneration in the index right eye and underwent intravitreal antivascular endothelial growth factor treatment for 5 years. A double-layer sign on in vivo OCT was correlated to calcified drusen on histology. Furthermore, hyperfluorescence on fluorescein angiography corresponded on histology to choroidal hypertransmission on OCT and retinal pigment epithelium atrophy above calcified drusen. CONCLUSION: A double-layer sign on OCT can represent nonneovascular subretinal pigment epithelium material including wide and flat calcific nodules. Furthermore, hyperfluorescence on FA, among different origins, can be due to a window defect corresponding to retinal pigment epithelium atrophy, which can be confirmed with OCT. Clinicopathological correlation using high-resolution histology can demonstrate the fine details available to clinical decision making through currently available in vivo OCT imaging.

Histology of Type 3 Macular Neovascularization and Microvascular Anomalies in Treated Age-Related Macular Degeneration: A Case Study.

Purpose: To investigate intraretinal neovascularization and microvascular anomalies by correlating in vivo multimodal imaging with corresponding ex vivo histology in a single patient. Design: A case study comprising clinical imaging from a community-based practice, and histologic analysis at a university-based research laboratory (clinicopathologic correlation). Participants: A White woman in her 90s treated with numerous intravitreal anti-VEGF injections for bilateral type 3 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD). Methods: Clinical imaging comprised serial infrared reflectance, eye-tracked spectral-domain OCT, OCT angiography, and fluorescein angiography. Eye tracking, applied to the 2 preserved donor eyes, enabled the correlation of clinical imaging signatures with high-resolution histology and transmission electron microscopy. Main Outcome Measures: Histologic/ultrastructural descriptions and diameters of vessels seen in clinical imaging. Results: Six vascular lesions were histologically confirmed (type 3 MNV, n = 3; deep retinal age-related microvascular anomalies [DRAMAs], n = 3). Pyramidal (n = 2) or tangled (n = 1) morphologies of type 3 MNV originated at the deep capillary plexus (DCP) and extended posteriorly to approach without penetrating persistent basal laminar deposit. They did not enter the subretinal pigment epithelium (RPE)-basal laminar space or cross the Bruch membrane. Choroidal contributions were not found. The neovascular complexes included pericytes and nonfenestrated endothelial cells, within a collagenous sheath covered by dysmorphic RPE cells. Deep retinal age-related microvascular anomaly lesions extended posteriorly from the DCP into the Henle fiber and the outer nuclear layers without evidence of atrophy, exudation, or anti-VEGF responsiveness. Two DRAMAs lacked collagenous sheaths. External and internal diameters of type 3 MNV and DRAMA vessels were larger than comparison vessels in the index eyes and in aged normal and intermediate AMD eyes. Conclusions: Type 3 MNV vessels reflect specializations of source capillaries and persist during anti-VEGF therapy. The collagenous sheath of type 3 MNV lesions may provide structural stabilization. If so, vascular characteristics may be useful in disease monitoring in addition to fluid and flow signal detection. Further investigation with longitudinal imaging before exudation onset will help determine if DRAMAs are part of the type 3 MNV progression sequence. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Impact of the Aging Lens and Posterior Capsular Opacification on Quantitative Autofluorescence Imaging in Age-Related Macular Degeneration.

Purpose: The purpose of this study was to investigate quantitative autofluorescence (qAF8) in patients with and without early or intermediate age-related macular degeneration (AMD); to determine the impact of the aged crystalline lens and posterior capsular opacification (PCO). Methods: In phakic and pseudophakic eyes ≥60 years, AMD status was determined by the Beckman system. PCO presence and severity was extracted from clinical records. qAF8 was calculated using custom FIJI plugins. Differences in qAF8, stratified by lens status, PCO severity, and AMD status, were analyzed using generalized estimating equations. Results: In 210 eyes of 115 individuals (mean age = 75.7 ± 6.6 years), qAF8 was lower in intermediate AMD compared to early AMD (P = 0.05). qAF8 did not differ between phakic and pseudophakic eyes (P = 0.8909). In phakic (n = 83) and pseudophakic (n = 127) eyes considered separately, qAF8 did not differ by AMD status (P = 0.0936 and 0.3494, respectively). Qualitative review of qAF images in phakic eyes illustrated high variability. In pseudophakic eyes, qAF8 did not differ with PCO present versus absent (54.5% vs. 45.5%). Review of implanted intraocular lenses (IOLs) revealed that 43.9% were blue-filter IOLs. Conclusions: qAF8 was not associated with AMD status, up to intermediate AMD, considering only pseudophakic eyes to avoid noisy images in phakic eyes. In pseudophakic eyes, qAF8 was not affected by PCO. Because blue-filter IOLs may reduce levels of exciting light for qAF8, future studies investigating qAF in eyes with different IOL types are needed. Translational Relevance: To reduce variability in observational studies and clinical trials requiring qAF8, pseudophakic participants without blue-filter IOLs or advanced PCO should be preferentially enrolled.

Correlation of Optical Coherence Tomography Angiography of Type 3 Macular Neovascularization With Corresponding Histology.

Importance: By validating optical coherence tomography angiography (OCTA) in the analysis of type 3 macular neovascularization secondary to age-related macular degeneration, the overall value of clinical OCTA for disease observation, diagnosis, and staging is increased. Objective: To assess the association of in vivo OCTA of type 3 macular neovascularization secondary to age-related macular degeneration with corresponding ex vivo histology. Design, Setting, and Participants: This study included clinical imaging, laboratory microscopy, and eye-tracked clinicopathologic correlation of a single case from a community-based practice evaluated at a university-based research laboratory from 2014 to 2019. Exposures: Infrared reflectance and eye-tracked spectral-domain OCTA clinical imaging was correlated with ex vivo high-resolution histologic images of the preserved donor eye. Eye tracking, applied to the donor eye, enabled identification of histologic features corresponding with clinical OCTA signatures. Projection artifact removal based on 2-dimensional vessel-shape estimation and a Gaussian blur filter demonstrated a robust preservation of neovascular flow signal. Main Outcomes and Measures: Histology findings associated with clinical OCTA signatures. Three-dimensional view of neovascularization via video. Results: A White woman in her 90s with type 3 neovascularization secondary to age-related macular degeneration was treated with 37 intravitreal injections of ranibizumab and aflibercept in the right eye. The index lesion displayed a drusenoid pigment epithelium detachment, characteristic of type 3 neovascularization. OCTA decorrelation signal in the index lesion corresponded in histology to a collagen-ensheathed vascular complex contacting basal laminar deposit that outlasted the retinal pigment epithelium. The subretinal pigment epithelium-basal laminar space contained calcified material and glial processes. No connection between the choriocapillaris and this space was observed. Video showed a columnar tangle of flow signal in the outer nuclear layer, with inflow and outflow vessels connecting to the superficial artery and vein. Conclusions and Relevance: While this study presents only 1 case in which a vascular connection between subretinal pigment epithelium-basal laminar space and choriocapillaris was undetected, these results support the potential value of OCTA for diagnosis. OCTA decorrelation signal of type 3 neovascularization corresponded with intraretinal neovessels on histology. Projection artifact removal based on 2-dimensional vessel-shape estimation and Gaussian blur filter demonstrated their potential value for further use in OCTA decorrelation signal processing.

Correlation of in vivo/ex vivo imaging of the posterior eye segment.

BACKGROUND: For an understanding of the pathology of retinal diseases, direct comparisons of high-resolution in vivo retinal imaging and ex vivo histological preparations are desirable. MATERIAL AND METHODS: Multimodal in vivo and ex vivo imaging of a human donor eye with secondary alterations showing atrophic retina due to central retinal arterial occlusion. The subsequent correlation with the histological examination was carried out on identical tissue localizations. RESULTS: Appropriate custom-built retinal imaging devices facilitate in vivo and ex vivo correlations and the examination of human eye tissue and acquisition of retinal images, e.g. SD-OCT. The precise alignment of the tissue enables a histological analysis on identical sites. CONCLUSION: The direct correlation of clinical in vivo imaging with ex vivo imaging including histopathology can further enhance our understanding in the pathogenesis of retinal diseases; however, the proposed method is currently limited due to restricted availability of human donor tissue.

Characteristics of normal human retinal pigment epithelium cells with extremes of autofluorescence or intracellular granule count.

BACKGROUND: Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities. METHODS: RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation. RESULTS: Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell. CONCLUSIONS: In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscins role in aging and AMD further into perspective.

Quantitative Fundus Autofluorescence in the Developing and Maturing Healthy Eye.

Purpose: Quantitative fundus autofluorescence (QAF) enables comparisons of autofluorescence intensities among participants. While clinical QAF reports mostly focused on the healthy and diseased adult retina, only very limited data of QAF in the maturing eye are available. Here, we report QAF in a large cohort of healthy children. Methods: In this prospective monocentric cross-sectional study, 70 healthy Caucasian children (5-18 years) were multimodal imaged, including QAF and spectral domain optical coherence tomography. QAF and retinal thicknesses were measured at predefined locations (along horizontal meridian; Early Treatment Diabetic Retinopathy Study [ETDRS] grid) and correlated using custom written Fiji plugins. Standard retinae for different age groups were generated. Results: Fifty-three participants were included. QAF was low in childhood but increased steadily (P < 0.001), also at the fovea (P < 0.001), with no gender differences (P = 0.61). The QAF distribution was similar to adults showing highest values superior-temporally. At individual points, retinal thickness remained stable, while using the ETDRS pattern, the retinal pigment epithelium (RPE) thickness increased significantly with aging. Standard QAF retinae of age groups also showed an increase with aging. Conclusions: QAF can be reliably performed in young children. Function-structure correlation showed a thickening of the RPE and an increasing QAF with aging, probably related to the histologic low number of RPE autofluorescent granules at a younger age but further deposition of these granules during maturation. Standard retinae help to distinguish abnormal QAF in the diseased retina of age-matched patients. Translational Relevance: Our data bridge the gap between preclinical QAF and clinical data application and structural OCT correlation in children.

Quantitative Fundus Autofluorescence in Systemic Chloroquine/Hydroxychloroquine Therapy.

Purpose: To investigate the effect of systemic chloroquine/hydroxychloroquine (CQ/HCQ) on outer retinal health using quantitative fundus autofluorescence (QAF) imaging. Methods: For this prospective, cross-sectional study, 44 CQ/HCQ patients and 25 age-matched controls underwent multimodal retinal imaging including QAF (488 nm) and spectral-domain optical coherence tomography (SD-OCT) in addition to the recommended CQ/HCQ screening procedures. Custom written FIJI plugins enabled detailed QAF analysis and correlation with retinal thickness and comparison to the healthy controls. Results: Out of 44 patients, 29 (mean age 43.5 ± 12.2, range 22-59 years) exposed to CQ/HCQ (mean cumulative dose 724.2 ± 610.4 g, median 608.0 g, range 18.6-2171.0 g) met eligibility criteria. Four of these 29 patients had bull's-eye maculopathy (BEM). Mean QAF values were significantly higher in CQ/HCQ patients than in healthy controls. QAF increase started early after treatment onset, remained high even years after treatment cessation, and was not accompanied by pathologies in the other screening methods, including retinal thicknesses (except in BEM patients). Conclusions: QAF might be a useful tool in retinal imaging and in verifying systemic CQ/HCQ intake. The early onset and preserved high levels of QAF parallel findings of CQ deposition in the retina in animal models. Whether QAF can be used as a screening tool to detect early CQ/HCQ related maculopathy is the subject of long-term ongoing studies. Translation Relevance: Experimental QAF imaging in systemic CQ/HCQ therapy monitoring might be a useful tool to indicate the drug or its metabolites and to detect metabolic retinal changes.

[Correlation of in vivo/ex vivo imaging of the posterior eye segment]. / Vergleichende In-vivo­/Ex-vivo-Bildgebung des hinteren Augenabschnitts.

BACKGROUND: For an understanding of the pathology of retinal diseases, direct comparisons of high-resolution in vivo retinal imaging and ex vivo histological preparations are desirable. MATERIAL AND METHODS: Multimodal in vivo and ex vivo imaging of a human donor eye with secondary alterations showing atrophic retina due to central retinal arterial occlusion. The subsequent correlation with the histological examination was carried out on identical tissue localizations. RESULTS: Appropriate custom-built retinal imaging devices facilitate in vivo and ex vivo correlations and the examination of human eye tissue and acquisition of retinal images, e.g. SD-OCT. The precise alignment of the tissue enables a histological analysis on identical sites. CONCLUSION: The direct correlation of clinical in vivo imaging with ex vivo imaging including histopathology can further enhance our understanding in the pathogenesis of retinal diseases; however, the proposed method is currently limited due to restricted availability of human donor tissue.

Quantitative Fundus Autofluorescence: Advanced Analysis Tools.

Purpose: To use multimodal retinal images (including quantitative fundus autofluorescence [QAF]) for spectral-domain optical coherence tomography (SD-OCT)-based image registration and alignment. For each age decade of healthy adults, normative fine-grained QAF retinal maps are generated and advanced methods for QAF image analysis are applied. Methods: Multimodal retinal images were obtained from 103 healthy subjects (age 19-77 years; unremarkable retina/macula, age-appropriate clear optic media). Custom written FIJI plugins enabled: (1) determination of the fovea in SD-OCT and the edge of the optic disc in infrared (IR) images; (2) alignment and superimposition of multimodal retinal images based on foveal and optic disc position; (3) plotting of normative QAF retinal maps for each decade; and (4) comparison of individual retinas with normative retinas of different decades using newly introduced analysis patterns (QAF97, freehand tool). Results: SD-OCT based image registration enables easy image registration, alignment, and analysis of different modalities (QAF, IR, and SD-OCT here reported). In QAF, intensities significantly increase with age with two major inclines between the third/fourth and seventh/eighth decades. With aging, the parafoveal area of maximum QAF intensity slightly shifts from temporal-superior to temporal. Compared with standard QAF analysis, refined QAF analysis patterns reveal a more detailed analysis of QAF, especially in the diseased retina. Conclusions: Age-related QAF normative retinal maps can be used to directly compare and classify individual's QAF intensities. Advanced QAF analysis tools will further help to interpret autofluorescence changes in normal aging and in the diseased retina in a multimodal imaging setting. Translational Relevance: Advanced methods for QAF analysis link basic findings with clinical observations in normal aging and in the diseased macula.

Bone Mass, Bone Microstructure and Biomechanics in Patients with Hand Osteoarthritis.

The impact of primary hand osteoarthritis (HOA) on bone mass, microstructure, and biomechanics in the affected skeletal regions is largely unknown. HOA patients and healthy controls (HCs) underwent high-resolution peripheral quantitative computed tomography (HR-pQCT). We measured total, trabecular, and cortical volumetric bone mineral densities (vBMDs), microstructural attributes, and performed micro-finite element analysis for bone strength. Failure load and scaled multivariate outcome matrices from distal radius and second metacarpal (MCP2) head measurements were analyzed using multiple linear regression adjusting for age, sex, and functional status and reported as adjusted Z-score differences for total and direct effects. A total of 105 subjects were included (76 HC: 46 women, 30 men; 29 HOA: 23 women, six men). After adjustment, HOA was associated with significant changes in the multivariate outcome matrix from the MCP2 head (p < .001) (explained by an increase in cortical vBMD (Δz = 1.07, p = .02) and reduction in the trabecular vBMD (Δz = -0.07, p = .09). Distal radius analysis did not show an overall effect of HOA; however, there was a gender-study group interaction (p = .044) explained by reduced trabecular vBMD in males (Δz = -1.23, p = .02). HOA was associated with lower failure load (-514 N; 95%CI, -1018 to -9; p = 0.05) apparent in males after adjustment for functional status. HOA is associated with reduced trabecular and increased cortical vBMD in the MCP2 head and a reduction in radial trabecular vBMD and bone strength in males. Further investigations of gender-specific changes of bone architecture in HOA are warranted. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.