Choroidal vascular alterations evaluated by ultra-widefield indocyanine green angiography in central serous chorioretinopathy.

PURPOSE: This study aims to evaluate choroidal vascular alterations in patients with central serous chorioretinopathy (CSC) using ultra-widefield (UWF) indocyanine green angiography (ICGA). METHODS: This was a retrospective case-control study conducted at a single tertiary eye center. In total, 36 eyes in patients with either unilateral (24 patients) or bilateral (six patients) treatment-naïve CSC and 30 eyes in 24 age-matched controls were evaluated. The number of quadrants with vortex vein engorgement on UWF ICGA was evaluated. Dilated choroidal vessels affecting the macula were regarded as extended vortex vein engorgement. Choroidal vascular hyperpermeability (CVH) area on late-phase ICGA was quantified using stereographic projection. The parameters were compared with clinical and optical coherence tomographic findings. RESULTS: Eyes with CSC had larger CVH area, thicker choroid, and more quadrants with vortex vein engorgement and extended vortex vein engorgement compared with control eyes (all P < 0.001). In patients with unilateral CSC, affected eyes had larger CVH area, thicker choroid, and more extended vortex vein engorgements compared with unaffected fellow eyes (all P < 0.001), but vortex vein engorgement did not significantly differ. CVH was significantly correlated with extended vortex vein engorgement (P < 0.001) and subfoveal choroidal thickness (P = 0.007). CONCLUSIONS: The increased number and binocular symmetry of engorged vortex veins suggest an anatomical predisposition for CSC. CVH area and extended vortex vein engorgement were indicators of choroidal outflow congestion. These parameters may serve as diagnostic clues or predictors of disease development in eyes with CSC.

Effects of refractive power on quantification using ultra-widefield retinal imaging.

BACKGROUND: Ultra-widefiled (UWF) retinal images include significant distortion when they are projected onto a two-dimensional surface for viewing. Therefore, many clinical studies that require quantitative analysis of fundus images have used stereographic projection algorithm, three-dimensional fundus image was mapped to a two-dimensional stereographic plane by projecting all relevant pixels onto a plane through the equator of the eye. However, even with this impressive algorithm, refractive error itself might affect the size and quality of images theoretically. The purpose of this study is to investigate the effects of refractive power on retinal area measurements (quantification) using UWF retinal imaging (Optos California; Dunfermline, Scotland, UK). METHODS: A prospective, interventional study comprised 50 healthy eyes. UWF images were acquired first without the use of a soft contact lens (CL) and then repeated with six CLs (+ 9D, +6D, +3D, -3D, -6D, and - 9D). Using stereographically projected UWF images, the optic disc was outlined by 15-17 points and quantified in metric units. We divided the subjects into three groups according to axial length: Groups A (22-24 mm), B (24-26 mm), and C (≥ 26 mm). The primary outcome was percentage change before and after use of the CLs. Secondary outcome was proportion of subjects with magnification effects, maximal changes > 10 %. RESULTS: The study population was 6, 28, and 16 eyes in each group. Overall changes for the measured area were not significantly different in the whole study population. Group C had a larger proportion of magnification effects compared to Groups A and B (50.0 %, 0 %, and 3.6 %, P = 0.020). Measured area with plus lenses was significantly higher in Group C (P < 0.001). CONCLUSIONS: The use of CLs might affect quantification of eyes with long axial length when using UWF images. Ophthalmologists should consider refractive error when measuring area in long eyes.

Distribution of peripheral lesions identified by mydriatic ultra-wide field fundus imaging in diabetic retinopathy.

PURPOSE: To analyze the distribution of diabetic retinopathy (DR) lesions in an Indian population using ultra-wide field (UWF) fundus imaging. METHODS: Seven hundred fifteen subjects (1406 eyes) with diabetic retinopathy in India were enrolled in this multicenter, prospective, observational study using UWF pseudocolor imaging with Optos Daytona Plus (Optos plc, Dunfermline, Scotland, UK). Images were transmitted to Doheny Image Reading Center, Los Angeles, CA, for grading. The ETDRS grid was overlaid on stereographic projections of UWF images, and images were graded independently by 2 masked graders. Lesion distribution was graded as predominantly central (PCL) or predominantly peripheral (PPL) according to previous criteria, considering both lesion number and area. An image was graded as PPL if > 50% of the lesion area was seen in at least one peripheral field as compared with the corresponding ETDRS field. Diabetic retinopathy severity was also assessed based on the International Classification of Diabetic Retinopathy (ICDR) grading scale. The main outcome measures were lesion distribution (PPL versus PCL): overall and within specific fields in eyes with various grades of DR. RESULTS: Lesion distribution was rated to be PPL in 37% of eyes and PCL in 63% of eyes (P < 0.003). The frequency of a PPL distribution varied significantly across all ICDR severity levels, with frequencies of mild non-proliferative DR (NPDR) (30.9%), moderate NPDR (40.3%), severe NPDR (38.5%) and PDR (34.9%), P = 0.005. When assessing which individual fields were rated to show a PPL distribution, the frequency was greatest in field 4 and least in field 7. For any grade of DR, temporal fields showed the greatest PPL frequency, followed in order by the superior, inferior, and nasal fields (P < 0.001). Only 3.5% of eyes showed PPL distribution in all five peripheral fields. CONCLUSIONS: One-third of the UWF images showed a PPL distribution in this cohort with the temporal quadrant having the widest distribution of PPL. As the PPL distribution varied significantly between various grades of DR, UWF imaging may prove to be important for screening of referral warranted retinopathy.

SEVERITY OF DIABETIC MACULAR EDEMA CORRELATES WITH RETINAL VASCULAR BED AREA ON ULTRA-WIDE FIELD FLUORESCEIN ANGIOGRAPHY: DAVE Study.

PURPOSE: To quantify retinal nonperfusion area and retinal vascular bed area (RVBA) in mm on ultra-widefield fluorescein angiography in eyes with diabetic macular edema (DME) and explore their relationship with the severity of DME. METHODS: Prospective, observational case series. Baseline ultra-widefield fluorescein angiography images of 40 eyes from 29 patients with treatment-naive DME who participated in the DAVE study (NCT01552408) were stereographically projected at Doheny Image Reading Center. The retinal vasculature was automatically extracted to calculate RVBA. Nonperfusion area was manually delineated by two masked certified graders. Retinal vascular bed area and nonperfusion area were computed in mm automatically by adjusting for peripheral distortion and then correlated with the severity of DME. RESULTS: The global RVBA for the entire retina in eyes with DME was increased compared with healthy controls (54.7 ± 16.6 mm vs. 37.2 ± 9.9 mm, P < 0.001) and correlated with the severity of DME (P < 0.05). Retinal ischemia (nonperfusion area) was nonuniformly distributed and not related to DME extent (P > 0.05). CONCLUSION: Eyes with DME have an increased RVBA compared with healthy controls. The severity of DME appears to be related to global RVBA, but not to retinal ischemia.

Hemorrhage and/or Microaneurysm Severity and Count in Ultrawide Field Images and Early Treatment Diabetic Retinopathy Study Photography.

OBJECTIVE: To evaluate detection of hemorrhage and/or microaneurysm (H/Ma) using ultrawide field (UWF) retinal imaging as compared with standard Early Treatment Diabetic Retinopathy Study (ETDRS) 7-field photographs (ETDRS photos). DESIGN: Single-site comparative study of UWF images and ETDRS photos. PARTICIPANTS: One hundred twenty-six eyes of 69 patients with no diabetic retinopathy (DR) or mild or moderate nonproliferative DR (NPDR). METHODS: Stereoscopic 200° UWF images and stereoscopic 35mm 30° 7-field color photographs were acquired on the same visit. Images were graded for severity and distribution of H/Ma. H/Mas were counted in ETDRS fields 2 to 7 in both ETDRS photos and UWF images. H/Mas in the UWF peripheral fields were also counted. MAIN OUTCOME MEASURES: Kappa (κ) and weighted κ statistics for agreement. Number of H/Ma within and outside ETDRS fields identified in UWF images and ETDRS photos. RESULTS: Distribution of DR severity by ETDRS photos was 24 (19.0%) no DR, 48 (38.1%) mild NPDR, and 54 (42.9%) moderate NPDR. A total of 748 of 756 fields (98.9%) were gradable for H/Mas on ETDRS photos and UWF images. Simple κ/weighted κ statistics for severity of H/Ma: all fields 0.61/0.69, field 2 0.70/0.77, field 3 0.62/0.73, field 4 0.50/0.62, field 5 0.54/0.65, field 6 0.64/0.70, and field 7 0.58/0.63 with overall exact agreement in 81.3% and within 1 step in 97.9% of fields. A greater proportion of fields was graded a more severe H/Ma level in UWF images than in the corresponding ETDRS photos (UWF: 12.7% vs. ETDRS: 6.5%). Evaluating comparable areas in UWF images and ETDRS photos (fields 2-7), a mean of 42.8 H/Mas were identified using ETDRS photos and 48.8 in UWF images (P = 0.10). An additional mean of 21.3 H/Mas (49.8% increase, P < 0.0001) were identified in the peripheral fields of the UWF images. CONCLUSIONS: There is good to excellent agreement between UWF images and ETDRS photos in determining H/Ma severity, with excellent correlation of H/Ma counts within ETDRS photo fields. UWF peripheral fields identified 49.8% more H/Ma, suggesting a more severe H/Ma in 12.7% of eyes. Given the additional lesions detected in peripheral fields and the known risks associated with H/Ma and peripheral lesions, quantification of H/Ma using UWF images may provide a more accurate representation of DR disease activity and potential greater accuracy in predicting DR progression.

Ultra-widefield Imaging of the Peripheral Retinal Vasculature in Normal Subjects.

PURPOSE: To establish the extent of the peripheral retinal vasculature in normal eyes using ultra-widefield (UWF) fluorescein angiography. DESIGN: Prospective, observational study. PARTICIPANTS: Fifty-nine eyes of 31 normal subjects, stratified by age, with no evidence of ocular disease in either eye by history and ophthalmoscopic examination. METHODS: Ultra-widefield fluorescein angiographic images were captured centrally and with peripheral steering using the Optos 200Tx (Optos, Dunfermline, United Kingdom). Images obtained at different gaze angles were montaged and corrected for peripheral distortion using a stereographic projection method to provide a single image for grading of the peripheral edge of the visible vasculature. The border of the vascularized retina was expressed as a radial surface distance from the center of the optic disc. The vascularized area was calculated based on this mean peripheral border position for each quadrant. MAIN OUTCOME MEASURES: Mean distance (mm) from the center of optic disc to the peripheral vascular border. RESULTS: In normal eyes, the mean radial surface distance from the center of the optic disc to the peripheral edge of the visible vasculature was 20.3±1.4 mm and the mean area of normal perfused retina was 977.0 mm(2). There was no significant difference between right and left eyes or between male and female participants. However, the distance to the periphery differed depending on the quadrant, with temporal (22.5±0.9 mm) being larger than inferior (20.4±1.7 mm) being larger than superior (19.2±1.5 mm) being larger than nasal (17.4±0.9 mm; P < 0.001) for all interquadrant comparisons. Interestingly, the distances to the perfused vascular border were significantly shorter in older individuals (≥60 years) than in younger subjects. CONCLUSIONS: Ultra-widefield fluorescein angiography is an important tool for studying the extent of peripheral retinal vasculature. With the increasing use of UWF imaging to evaluate and manage patients with retinal vascular disease, the normative data from this study may provide a useful reference when assessing the pathologic significance of findings in the setting of disease.

Diabetic Retinopathy Severity and Peripheral Lesions Are Associated with Nonperfusion on Ultrawide Field Angiography.

PURPOSE: To assess whether the presence of peripheral nonperfusion on ultrawide field (UWF) fluorescein angiography (FA) is associated with diabetic retinopathy (DR) severity and the presence of predominantly peripheral lesions (PPLs). DESIGN: Single-site, cross-sectional, retrospective study. PARTICIPANTS: Sixty-eight eyes of 37 diabetic subjects with or without DR and no history of prior panretinal laser photocoagulation. METHODS: Both 200° UWF images and UWF FA images were acquired at the same visit. Early Treatment Diabetic Retinopathy Study (ETDRS) templates were overlaid digitally based on disc and macula location onto stereographically projected UWF images. Images were evaluated for the presence of PPLs, defined as more than 50% of the graded lesion located outside the ETDRS field in each of the 5 extended fields. The UWF-FA images were evaluated by 2 masked, independent graders for extent of retinal nonperfusion area (NPA) and nonperfusion index (NPI; nonperfused/total gradable area). MAIN OUTCOME MEASURES: Association of NPA and NPI with DR severity and presence of PPLs. RESULTS: Distribution of DR severity was as follows: no DR, 8.8% eyes; mild nonproliferative DR (NPDR), 17.6%; moderate NPDR, 32.4%; severe NPDR, 17.6%; proliferative DR (PDR), 19.1%; and high-risk PDR, 4.4%; with PPL present in 61.8%. There was strong intragrader (r = 0.95) and intergrader (r = 0.86) agreement for NPA. Presence of PPLs was associated with increased NPA (191.8 mm(2) vs. 306.1 mm(2); P = 0.0019) and NPI (0.25 vs. 0.43; P = 0.0003). These relationships remained significant after adjusting for DR severity and diabetes duration. In eyes without PDR (n = 52), increasing NPA and NPI was associated with worsening DR (NPA, P = 0.001; NPI, P = 0.0003). NPA and NPI were not associated with clinically significant macular edema (NPA, P = 0.99; NPI, P = 0.67), nor correlated with visual acuity (NPA, r = 0.14, P = 0.23; NPI, r = 0.24, P = 0.05). CONCLUSIONS: Following a standardized protocol, the evaluation of UWF FA for NPA and NPI is reproducible. Both parameters are correlated highly with the presence of PPLs and DR severity. Given that the presence and extent of PPLs have been associated with increased risks of DR progression, the clinical identification of PPLs may reflect closely the extent of nonperfusion and ischemia, thus accounting for the increased risk of progression.

Baseline retinal vascular bed area on ultra-wide field fluorescein angiography correlates with the anatomical outcome of diabetic macular oedema to ranibizumab therapy: two-year analysis of the DAVE Study.

PURPOSE: To determine the relationship between baseline retinal non-perfusion area (NPA) and retinal vascular bed area (RVBA) on ultra-wide field fluorescein angiography (UWF FA) and long-term response to intravitreal ranibizumab therapy in diabetic macular oedema (DMO). METHODS: A post-hoc, 2-year observational case series. Baseline UWF FA images (Optos 200Tx) of 40 eyes from 29 patients with diabetes mellitus and treatment naïve DMO in the DAVE (NCT01552408) study were montaged and stereographically projected at the Doheny Image Reading Center to adjust for peripheral distortion. The retinal vasculature was automatically extracted to calculate RVBA. NPA was manually delineated by two masked certified graders. RVBA and NPA were computed in mm2 automatically by adjusting for peripheral distortion and then correlated with the severity of DMO. RESULTS: While global NPA at baseline was not correlated to retinal thickness measurements, baseline NPA in the superior retina was associated with the macular volume (MV) improvement (P = 0.022). Multivariate analysis revealed a smaller RVBA at baseline was correlated with a better MV outcome at two-year follow-up after adjusting for confounding factors (P = 0.049). CONCLUSION: Eyes with smaller baseline RVBA appear to have a better long-term anatomic outcome of DMO.

EnzML: multi-label prediction of enzyme classes using InterPro signatures.

BACKGROUND: Manual annotation of enzymatic functions cannot keep up with automatic genome sequencing. In this work we explore the capacity of InterPro sequence signatures to automatically predict enzymatic function. RESULTS: We present EnzML, a multi-label classification method that can efficiently account also for proteins with multiple enzymatic functions: 50,000 in UniProt. EnzML was evaluated using a standard set of 300,747 proteins for which the manually curated Swiss-Prot and KEGG databases have agreeing Enzyme Commission (EC) annotations. EnzML achieved more than 98% subset accuracy (exact match of all correct Enzyme Commission classes of a protein) for the entire dataset and between 87 and 97% subset accuracy in reannotating eight entire proteomes: human, mouse, rat, mouse-ear cress, fruit fly, the S. pombe yeast, the E. coli bacterium and the M. jannaschii archaebacterium. To understand the role played by the dataset size, we compared the cross-evaluation results of smaller datasets, either constructed at random or from specific taxonomic domains such as archaea, bacteria, fungi, invertebrates, plants and vertebrates. The results were confirmed even when the redundancy in the dataset was reduced using UniRef100, UniRef90 or UniRef50 clusters. CONCLUSIONS: InterPro signatures are a compact and powerful attribute space for the prediction of enzymatic function. This representation makes multi-label machine learning feasible in reasonable time (30 minutes to train on 300,747 instances with 10,852 attributes and 2,201 class values) using the Mulan Binary Relevance Nearest Neighbours algorithm implementation (BR-kNN).

Automatically identifying and annotating mouse embryo gene expression patterns.

MOTIVATION: Deciphering the regulatory and developmental mechanisms for multicellular organisms requires detailed knowledge of gene interactions and gene expressions. The availability of large datasets with both spatial and ontological annotation of the spatio-temporal patterns of gene expression in mouse embryo provides a powerful resource to discover the biological function of embryo organization. Ontological annotation of gene expressions consists of labelling images with terms from the anatomy ontology for mouse development. If the spatial genes of an anatomical component are expressed in an image, the image is then tagged with a term of that anatomical component. The current annotation is done manually by domain experts, which is both time consuming and costly. In addition, the level of detail is variable, and inevitably errors arise from the tedious nature of the task. In this article, we present a new method to automatically identify and annotate gene expression patterns in the mouse embryo with anatomical terms. RESULTS: The method takes images from in situ hybridization studies and the ontology for the developing mouse embryo, it then combines machine learning and image processing techniques to produce classifiers that automatically identify and annotate gene expression patterns in these images. We evaluate our method on image data from the EURExpress study, where we use it to automatically classify nine anatomical terms: humerus, handplate, fibula, tibia, femur, ribs, petrous part, scapula and head mesenchyme. The accuracy of our method lies between 70% and 80% with few exceptions. We show that other known methods have lower classification performance than ours. We have investigated the images misclassified by our method and found several cases where the original annotation was not correct. This shows our method is robust against this kind of noise. AVAILABILITY: The annotation result and the experimental dataset in the article can be freely accessed at http://www2.docm.mmu.ac.uk/STAFF/L.Han/geneannotation/. CONTACT: l.han@mmu.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clinical significance of metabolic quantification for retinal nonperfusion in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by microvascular changes including ischemia. Degradation and metabolic changes of various retinal cells occur during ischemia. Ischemic region containing more cells will lead to greater metabolic impairment. We analyzed the non-perfusion region (NPR) by integrating histologic mapping with ultra-widefield fluorescein angiography (UWF FA) images. We also investigated the correlations of the weighted ischemic index (ISI) considering the regional distribution of retinal cells with cytokines, macular edema (ME), and neovascularization (NV). In this study, 32 patients with treatment-naïve DR and 21 age-matched control participants were included. The difference between the non-weighted and weighted ISI of NPR with leakage was greatest at the posterior region. The weighted ISI of NPR with leakage was correlated with MCP-1, IL-8, IL-6, PlGF, and VEGF-A levels, while the non-weighted ISI of NPR with leakage was correlated with IL-8 and IL-6 levels. The presence of baseline ME or NV in patients with DR was associated with the weighted ISI, with a stronger association when cones and rods were weighted. The weighted ISI reflecting both metabolic activity and cell distribution demonstrated a better correlation with clinical features and was more valuable in NPR with leakage than non-weighted ISI, which previous studies conventionally used.

Multi-modal retinal scanning to measure retinal thickness and peripheral blood vessels in multiple sclerosis.

Our purpose was to investigate changes to the retina in multiple sclerosis (MS) using established and novel modes of retinal image acquisition and analysis. 72 participants with MS and 80 healthy volunteers underwent retinal scanning with optical coherence tomography (OCT) and ultra-widefield (UWF) scanning laser ophthalmoscopy (SLO), over a two-year period. Changes in retinal nerve fibre layer (RNFL) thickness, macular volume and retinal blood vessel diameter were measured and parameters were then tested for associations with MS. Measurements from OCT showed that individuals with MS had a thinner RNFL and reduced macular volume when compared to healthy volunteers. On UWF images, participants with MS had reduced arterial widths in the inferior nasal quadrant of both eyes and reduced venous widths in the inferior nasal quadrant of right eyes. Longitudinal analysis showed that participants with MS had an accelerated annual rate of RNFL thinning in several regions of the retina. In conclusion, the assessment of OCT showed thinning of the RNFL and macula in concordance with previous reports on MS, while analysis of blood vessels in the retinal periphery from UWF-SLO images revealed novel changes.

Retinal vascular bed area on ultra-wide field fluorescein angiography indicates the severity of diabetic retinopathy.

AIMS: To quantify retinal vascular bed area (RVBA) in square millimetres on stereographically projected ultra-wide field (UWF) fluorescein angiography (FA) in eyes with diabetic retinopathy (DR). METHODS: A prospective, observational study. Baseline Optos 200Tx UWF FA images of 80 eyes with DR from the DAVE (NCT01552408) and RECOVERY (NCT02863354) studies were stereographically projected at the Doheny Image Reading Center to adjust for peripheral distortion. The early-phase FA frame was used to extract the retinal vasculature as a mask for calculating RVBA. The pixels of the retinal vasculature were automatically computed in square millimetres using manufacturer-provided software. RESULTS: Eighteen of 80 diabetic eyes were excluded because image quality and contrast were insufficient for automatic extraction of the retinal vasculature from the background fluorescence. The remaining 62 eyes were included in the final analysis. In comparison with age-matched and sex-matched normal controls, eyes with DR had a higher global RVBA for the entire retina (p<0.001), and RVBA correlated with DR severity (p<0.001), with a higher RVBA in eyes with proliferative DR (66.1±16.2 mm2) than in those with non-proliferative DR (56.2±16.6 mm2) or in normal controls (37.2±9.9 mm2). This tendency was also present in the posterior retina and mid-periphery but absent in the far-periphery. RVBA did not correlate with retinal ischaemia (p>0.05). CONCLUSIONS: Eyes with DR harboured a larger global RVBA for the entire retina than normal controls, and RVBA appeared to indicate DR severity. However, this biomarker was not observed to be a good indicator of retinal ischaemia.

A user-friendly web portal for T-Coffee on supercomputers.

BACKGROUND: Parallel T-Coffee (PTC) was the first parallel implementation of the T-Coffee multiple sequence alignment tool. It is based on MPI and RMA mechanisms. Its purpose is to reduce the execution time of the large-scale sequence alignments. It can be run on distributed memory clusters allowing users to align data sets consisting of hundreds of proteins within a reasonable time. However, most of the potential users of this tool are not familiar with the use of grids or supercomputers. RESULTS: In this paper we show how PTC can be easily deployed and controlled on a super computer architecture using a web portal developed using Rapid. Rapid is a tool for efficiently generating standardized portlets for a wide range of applications and the approach described here is generic enough to be applied to other applications, or to deploy PTC on different HPC environments. CONCLUSIONS: The PTC portal allows users to upload a large number of sequences to be aligned by the parallel version of TC that cannot be aligned by a single machine due to memory and execution time constraints. The web portal provides a user-friendly solution.

Macular Microvascular Changes and Their Correlation With Peripheral Nonperfusion in Branch Retinal Vein Occlusion.

PURPOSE: To investigate the correlation between macular microvascular alterations on optical coherence tomography angiography (OCTA) and retinal ischemia on ultra-widefield fluorescein angiography (UWF FA) in eyes with branch retinal vein occlusion (BRVO). DESIGN: Cross-sectional study. METHODS: This prospective study was performed from September 2019 to June 2020 at Yeungnam University Medical Center. We included 60 patients with treatment-naïve BRVO. Two independent, masked graders analyzed OCTA parameters, including vessel density, skeletal density, and fractal dimension (FD), and UWF FA parameters, including retinal nonperfusion area (NPA) and ischemic index (ISI), from various concentric regions (perimacular region, 0.5-3 mm radius; near-peripheral region, 3-10 mm; midperipheral region, 10-15 mm; far-peripheral region, >15 mm). A repeated-measures analysis of variance test and a paired t test were performed for inter-visit and inter-regional comparisons, and Pearson correlation coefficient and multivariate regression analyses were performed to examine the correlation between UWF FA and OCTA parameters. RESULTS: The OCTA parameters from both the superficial and deep capillary plexuses (DCP) were significantly correlated with NPA and ISI in all concentric regions. Even after adjusting for several covariates, all OCTA parameters revealed a significant association with ISI on UWF FA. Moreover, OCTA parameters from DCP were significantly correlated with concentrations of placental growth factor and vascular endothelial growth factor. Although all OCTA parameters achieved excellent results of area under the curve (AUC) > 0.9 for detecting severe retinal ischemia, defined as ISI >10%, FD reduction in DCP was the most reliable parameter (AUC = 0.948, P < .001), and 5.39% was the best cut-off point for predicting ISI > 10%. CONCLUSIONS: OCTA is a useful noninvasive tool not only for evaluation of macular microvasculature but for supposition of peripheral nonperfusion in eyes with BRVO.

Relationship between distribution and severity of non-perfusion and cytokine levels and macular thickness in branch retinal vein occlusion.

We aimed to investigate the relationship between non-perfusion on ultra-widefield angiography (UWF FA) and aqueous cytokine levels and central macular thickness (CMT) in eyes with branch retinal vein occlusion (BRVO). Thirty-five eyes with treatment-naïve BRVO were included. Non-perfusion area (NPA) for partial and complete ischemia was manually segmented and the ischemic index (ISI) for each was calculated using stereographically projected UWF FA for four different retinal zones. Partial and complete ischemia had different regional predominance. Partial ischemia was predominant in the posterior regions, while complete ischemia was predominant in the periphery. And partial ischemic area, located posterior to far periphery, showed significant correlation with central macular thickness and concentrations of angiogenic and inflammatory cytokines, while complete ischemic area showed no correlation with any of the parameters. Taken together, partial but not complete ischemia, particularly in the more posterior retina, was associated with higher cytokine levels and more severe macular edema in eyes with BRVO. These findings would help us to better understand the different clinical significance of ischemia in BRVO depending on the severity and regional distribution.

Peripheral extent of the choroidal circulation by ultra-widefield indocyanine green angiography in healthy eyes.

AIM: To analyse the peripheral extent of choroidal circulation using ultra-widefield (UWF) indocyanine green angiography (ICGA) in healthy eyes. METHODS: UWF ICGA images of 55 eyes of 36 healthy subjects were captured using the Optos California (Optos, Dunfermline, United Kingdom) in this prospective observational study. Images were analysed to locate the peripheral extent of the visible choroidal circulation, and the boundary was marked in ImageJ (v1.52). Each pixel annotated as the border of the choroidal circulation was projected individually to its anatomically correct location on the three-dimensional model eye, and spherical trigonometry was applied (using the Optos software) to calculate its respective radial distance from the centre of the optic disc in metric units (corrected by stereographic projection) for each quadrant. RESULTS: The mean area of the peripheral extent was estimated to be 893.2 mm2 (95% CI: 844.2 to 942.3 mm2). The mean distance (range) of this boundary from optic nerve centre was 18.22 mm (95% CI: 14.0 to 23.14 mm). Multiple regression analysis with age, gender, axial length or ethnicity showed no relationship. There was excellent inter-grader reproducibility, with intraclass correlation coefficients of 0.95 (95% CI: 0.80 to 0.99, p<0.001) for distance and 0.99 (95% CI: 0.988 to 0.999, p<0.001) for area measurements. CONCLUSIONS: The peripheral choroidal boundary may be defined using UWF ICGA. Knowledge of the normal extent and its variability is essential to understand the impact of disease on the choroidal vasculature.

Retinal Vascular Caliber Association with Nonperfusion and Diabetic Retinopathy Severity Depends on Vascular Caliber Measurement Location.

PURPOSE: To evaluate the association of retinal nonperfusion and diabetic retinopathy (DR) severity with location of vascular caliber measurement using ultrawide field (UWF) imaging. DESIGN: Retrospective image review. PARTICIPANTS: Adults with diabetes mellitus. METHODS: All images from subjects with same-day UWF fluorescein angiography (FA) and color imaging were evaluated. Predominantly peripheral lesions (PPL) and DR severity were graded from UWF color images. Nonperfusion was quantified using UWF-FA in defined retinal regions [posterior pole (PP), mid-periphery (MP), far-periphery (FP)]. Retinal vessel calibers were measured at an optic disc centered inner and outer zone. MAIN OUTCOME MEASURES: Nonperfusion index (NPI) in the PP, MP and FP. Mean arteriole and venule diameter in the inner and outer zones. RESULTS: Two hundred eighty-five eyes of 193 patients (24.9% mild nonproliferative DR [NPDR], 22.8% moderate NPDR, 37.5% severe NPDR and 14.7% proliferative DR [PDR]) were reviewed. No significant associations between inner zone arteriolar diameter and retinal NPI overall or in any retinal region. In the outer zone, eyes with thinnest arteriolar calibers (quartile 1) were associated with a 1.7- to 2.4-fold nonperfusion increase across all retinal regions compared to the remaining eyes (P = 0.002 [PP] to 0.048 [FP]). In the outer zone, the percentage of eyes in the thinnest quartile of retinal arteriolar diameter increased with worsening DR severity (mild NPDR: 10% vs PDR: 31%, P = 0.007). This association was not observed when measured within the inner zone (P = 0.129). All venular caliber associations were not statistically significant when corrected for potentially confounding factors. Thinner outer zone retinal arteriolar caliber (quartile 1) was more common in eyes with PPL compared to eyes without PPL (34.1% vs 20.8%, P = 0.017) as were thicker outer venular calibers (quartile 4) (33% vs 21.3%, P = 0.036). Presence of PPL was associated with thinner outer zone arteriolar caliber (109.7 ± 26.5µm vs 123.0 ± 29.5µm, P = 0.001). CONCLUSIONS: The association of vascular caliber with nonperfusion and DR severity differs based upon the retinal location at which vascular caliber is measured. Peripheral arterial narrowing is associated with increasing nonperfusion, worsening DR severity and presence of PPL. In contrast, inner zone retinal arteriolar caliber is not associated with these findings.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles.

BACKGROUND: Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. RESULTS: A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. CONCLUSION: In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data.