Background/Aims To analyse the retest reliability of visual field indices and to describe patterns of visual field deficits in mesopic and dark-adapted two-colour fundus-controlled perimetry (FCP) in macular diseases. Methods Seventy-seven eyes (30 eyes with macular diseases and 47 normal eyes) underwent duplicate mesopic and dark-adapted two-colour FCP (Scotopic Macular Integrity Assessment, CenterVue). Non-weighted (mean defect, loss variance), variability-weighted (mean deviation, pattern standard deviation (PSD)) and graphical (cumulative defect (Bebie) curves) indices were computed. Reproducibility (coefficient of repeatability, CoR) of these indices was assessed. Cluster analysis was carried out to identify patterns of visual field deficits. Results The intrasession reproducibility was lower for the mean defect as compared with the mean deviation (CoR (dB) 2.67 vs 2.57 for mesopic, 1.71 vs 1.45 for dark-adapted cyan, 1.94 vs 1.87 for dark-adapted red testing) and lower for the square-root loss variance as compared with the PSD (CoR (dB) 1.48 vs 1.34, 0.77 vs 0.65, 1.23 vs 1.03). Hierarchical cluster analysis of the indices disclosed six patterns of visual field deficits (approximately unbiased P value>0.95) with varying degrees of global versus focal defect and rod versus cone dysfunction. These were also reflected by the cumulative defect curves. Conclusion FCP with mesopic and dark-adapted two-colour testing allows for reproducible assessment of different types of retinal sensitivity, whereby mean deviation and PSD exhibited the better retest reliability of the tested indices. Distinct patterns of retinal dysfunction can be identified using this setup, reflecting variable degrees of rod and cone dysfunction in different macular diseases. Dark-adapted two-colour FCP provides additional diagnostic information and allows for refined structure–function correlation in macular diseases.
Research on extracellular vesicles (EVs), both in general laboratory methods and in novel therapeutic approaches, is limited by the difficulty of obtaining large quantities of EVs. This is due to the fact that EVs are only present at low concentrations in most starting samples and common methods for concentrating EVs from large volumes have decisive disadvantages. In particular, ultracentrifugation (UC) and tangential flow filtration (TFF) are used in EV bulk production. However, while UC leads to low EV-yield with high levels of impurities and damaged EVs, TFF is suitable for enriching EVs from large volumes, but requires expensive equipment. In addition, both methods require pre-purification of the EV material and downstream EV isolation steps. To circumvent all these obstacles, we evaluated various methods for their applicability to EV mass purification, optimized both their implementation and the workflow, and thus developed the following sequential protocol for EV bulk production: First, a preclearing step is performed by 1µm filtration, followed by PEG precipitation to reduce total volume. Larger debris and apoptotic bodies are removed by centrifugation and filtration prior to enrichment and pre-purification of EVs by ultrafiltration or using the ExoEasyMaxi kit. Finally, EVs are purified by size exclusion chromatography. Since the availability of suitable starting material to obtain pure EVs is also a challenge, we compared different biological fluids, with urine proving to be the most suitable source. We determined the purity and quantity of the EV samples using nanotracking analysis, dynamic light scattering, phospholipid-, RNA- and protein-concentration measurement, electron microscopy and western blot.In summary, we have developed procedures to obtain about 1012 pure EVs in less than a day, with a handling time under 2 hours, and at a cost of approximately €24. This method should facilitate further scientific and technical developments in the field of EV biology.
Research on extracellular vesicles (EVs), both in general laboratory methods and in novel therapeutic approaches, is limited by the difficulty of obtaining large quantities of EVs. This is due to the fact that EVs are only present at low concentrations in most starting samples and common methods for concentrating EVs from large volumes have decisive disadvantages. In particular, ultracentrifugation (UC) and tangential flow filtration (TFF) are used in EV bulk production. However, while UC leads to low EV-yield with high levels of impurities and damaged EVs, TFF is suitable for enriching EVs from large volumes, but requires expensive equipment. In addition, both methods require pre-purification of the EV material and downstream EV isolation steps. To circumvent all these obstacles, we evaluated various methods for their applicability to EV mass purification, optimized both their implementation and the workflow, and thus developed the following sequential protocol for EV bulk production: First, a preclearing step is performed by 1 µm filtration, followed by PEG precipitation to reduce total volume. Larger debris and apoptotic bodies are removed by centrifugation and filtration prior to enrichment and pre-purification of EVs by ultrafiltration or using the ExoEasyMaxi kit. Finally, EVs are purified by size exclusion chromatography. Since the availability of suitable starting material to obtain pure EVs is also a challenge, we compared different biological fluids, with urine proving to be the most suitable source. We determined the purity and quantity of the EV samples using nanotracking analysis, dynamic light scattering, phospholipid-, RNA- and protein-concentration measurement, electron microscopy and western blot. In summary, we have developed procedures to obtain about 1012 pure EVs in less than a day, with a handling time under 2 h, and at a cost of approximately €24. This method should facilitate further scientific and technical developments in the field of EV biology.
To develop and evaluate a software tool for automated detection of focal hyperpigmentary changes (FHC) in eyes with intermediate age-related macular degeneration (AMD).Color fundus (CFP) and autofluorescence (AF) photographs of 33 eyes with FHC of 28 AMD patients (mean age 71 years) from the prospective longitudinal natural history MODIAMD-study were included. Fully automated to semiautomated registration of baseline to corresponding follow-up images was evaluated. Following the manual circumscription of individual FHC (four different readings by two readers), a machine-learning algorithm was evaluated for automatic FHC detection.The overall pixel distance error for the semiautomated (CFP follow-up to CFP baseline: median 5.7; CFP to AF images from the same visit: median 6.5) was larger as compared for the automated image registration (4.5 and 5.7; P < 0.001 and P < 0.001). The total number of manually circumscribed objects and the corresponding total size varied between 637 to 1163 and 520,848 pixels to 924,860 pixels, respectively. Performance of the learning algorithms showed a sensitivity of 96% at a specificity level of 98% using information from both CFP and AF images and defining small areas of FHC ("speckle appearance") as "neutral."FHC as a high-risk feature for progression of AMD to late stages can be automatically assessed at different time points with similar sensitivity and specificity as compared to manual outlining. Upon further development of the research prototype, this approach may be useful both in natural history and interventional large-scale studies for a more refined classification and risk assessment of eyes with intermediate AMD.Automated FHC detection opens the door for a more refined and detailed classification and risk assessment of eyes with intermediate AMD in both natural history and future interventional studies.
Purpose: To examine the topographic correlation between retinal morphology and retinal sensitivity by mesopic and scotopic fundus-controlled perimetry (FCP) in eyes with intermediate AMD. Methods: Thirty-five eyes from 32 patients (mean age 70.9 years) and 29 age-matched controls prospectively underwent spectral-domain optical coherence tomography (SD-OCT) imaging. Mesopic (Goldman III, 200 ms, 4–2 strategy) and scotopic (Goldman V, 200 ms, 4–2 strategy) FCP with a 56-stimulus point grid was performed in AMD patients with the MP-1S. Thickness values of different retinal layers were measured at each stimulus point and compared, topographically corresponding to values in controls of similar age for pointwise structural-functional analysis. Results: The overall mean sensitivity in patients was 16.9 ± 3.0 dB for mesopic and 14.0 ± 3.7 dB for scotopic testing. Within the central 4° of the macula, reduced mesopic and scotopic sensitivity values were found (P < 0.0001). These findings correlated to central increasing retinal pigment epithelium–drusen complex (RPEDC) thickness and central decreasing outer nuclear layer (ONL) and photoreceptor (PR)-segments thickness (P < 0.0001, respectively). Structure-function correlations revealed that a reduction of mesopic and scotopic sensitivity was associated with increasing thickness of the total retina and the RPEDC and a decrease of the ONL and the PR-segments (P < 0.001, respectively). Conclusions: Accumulation of sub-RPE material in patients with intermediate AMD is spatially associated to quantifiable structural alterations in various retinal layers and to corresponding retinal dysfunction. The topographic analysis of retinal thickness and retinal sensitivity will be helpful for a better understanding of the disease process and for the evaluation of new interventional approaches.
Importance One of the biggest challenges when using anti–vascular endothelial growth factor (VEGF) agents to treat retinopathy of prematurity (ROP) is the need to perform long-term follow-up examinations to identify eyes at risk of ROP reactivation requiring retreatment. Objective To evaluate whether an artificial intelligence (AI)–based vascular severity score (VSS) can be used to analyze ROP regression and reactivation after anti-VEGF treatment and potentially identify eyes at risk of ROP reactivation requiring retreatment. Design, Setting, and Participants This prognostic study was a secondary analysis of posterior pole fundus images collected during the multicenter, double-blind, investigator-initiated Comparing Alternative Ranibizumab Dosages for Safety and Efficacy in Retinopathy of Prematurity (CARE-ROP) randomized clinical trial, which compared 2 different doses of ranibizumab (0.12 mg vs 0.20 mg) for the treatment of ROP. The CARE-ROP trial screened and enrolled infants between September 5, 2014, and July 14, 2016. A total of 1046 wide-angle fundus images obtained from 19 infants at predefined study time points were analyzed. The analyses of VSS were performed between January 20, 2021, and November 18, 2022. Interventions An AI-based algorithm assigned a VSS between 1 (normal) and 9 (most severe) to fundus images. Main Outcomes and Measures Analysis of VSS in infants with ROP over time and VSS comparisons between the 2 treatment groups (0.12 mg vs 0.20 mg of ranibizumab) and between infants who did and did not receive retreatment for ROP reactivation. Results Among 19 infants with ROP in the CARE-ROP randomized clinical trial, the median (range) postmenstrual age at first treatment was 36.4 (34.7-39.7) weeks; 10 infants (52.6%) were male, and 18 (94.7%) were White. The mean (SD) VSS was 6.7 (1.9) at baseline and significantly decreased to 2.7 (1.9) at week 1 ( P &lt; .001) and 2.9 (1.3) at week 4 ( P &lt; .001). The mean (SD) VSS of infants with ROP reactivation requiring retreatment was 6.5 (1.9) at the time of retreatment, which was significantly higher than the VSS at week 4 ( P &lt; .001). No significant difference was found in VSS between the 2 treatment groups, but the change in VSS between baseline and week 1 was higher for infants who later required retreatment (mean [SD], 7.8 [1.3] at baseline vs 1.7 [0.7] at week 1) vs infants who did not (mean [SD], 6.4 [1.9] at baseline vs 3.0 [2.0] at week 1). In eyes requiring retreatment, higher baseline VSS was correlated with earlier time of retreatment (Pearson r = −0.9997; P &lt; .001). Conclusions and Relevance In this study, VSS decreased after ranibizumab treatment, consistent with clinical disease regression. In cases of ROP reactivation requiring retreatment, VSS increased again to values comparable with baseline values. In addition, a greater change in VSS during the first week after initial treatment was found to be associated with a higher risk of later ROP reactivation, and high baseline VSS was correlated with earlier retreatment. These findings may have implications for monitoring ROP regression and reactivation after anti-VEGF treatment.
