Optical Coherence Tomography Angiography of Volumetric Arteriovenous Relationships in the Healthy Macula and Their Derangement in Disease.

Purpose: To characterize relative arteriovenous connectivity of the healthy macula imaged by optical coherence tomography angiography (OCTA) using a new volumetric tool. Methods: OCTA volumes were obtained for 20 healthy controls (20 eyes). Two graders identified superficial arterioles and venules. We implemented a custom watershed algorithm to identify capillaries most closely connected to arterioles and venules by using the large vessels as seeds to flood the vascular network. We calculated ratios of arteriolar- to venular-connected capillaries (A/V ratios) and adjusted flow indices (AFIs) for superficial capillary plexuses (SCPs), middle capillary plexuses (MCPs), and deep capillary plexuses (DCPs). We also analyzed two eyes with proliferative diabetic retinopathy (PDR) and one eye with macular telangiectasia (MacTel) to evaluate the utility of this method in visualizing pathological vascular connectivity. Results: In healthy eyes, the MCP showed a greater proportion of arteriolar-connected vessels than the SCP and DCP (all P < 0.001). In the SCP, the arteriolar-connected AFI exceeded the venular-connected AFI, but this pattern reversed in the MCP and DCP, with higher venular-connected AFI (all P < 0.001). In PDR eyes, preretinal neovascularization originated from venules, whereas intraretinal microvascular abnormalities were heterogeneous, with some originating from venules and others representing dilated MCP capillary loops. In MacTel, diving SCP venules formed the epicenter of the outer retinal anomalous vascular network. Conclusions: Healthy eyes showed a higher MCP A/V ratio but relatively slower arteriolar vs. venular flow velocity in the MCP and DCP, which may explain deep retinal vulnerability to ischemia. In eyes with complex vascular pathology, our connectivity findings were consistent with histopathologic studies.

Deep Capillary Nonperfusion on OCT Angiography Predicts Complications in Eyes with Referable Nonproliferative Diabetic Retinopathy.

OBJECTIVE: To evaluate the ability of capillary nonperfusion parameters on OCT angiography (OCTA) to predict the development of clinically significant outcomes in eyes with referable nonproliferative diabetic retinopathy (NPDR). DESIGN: Prospective longitudinal observational study. SUBJECTS: In total, 59 patients (74 eyes) with treatment-naive moderate and severe (referable) NPDR. METHODS: Patients were imaged with OCTA at baseline and then followed-up for 1 year. We evaluated 2 OCTA capillary nonperfusion metrics, vessel density (VD) and geometric perfusion deficits (GPDs), in the superficial capillary plexus, middle capillary plexus (MCP), and deep capillary plexus (DCP). We compared the predictive accuracy of baseline OCTA metrics for clinically significant diabetic retinopathy (DR) outcomes at 1 year. MAIN OUTCOME MEASURES: Significant clinical outcomes at 1 year, defined as 1 or more of the following-vitreous hemorrhage, center-involving diabetic macular edema, and initiation of treatment with pan-retinal photocoagulation or anti-VEGF injections. RESULTS: Overall, 49 patients (61 eyes) returned for the 1-year follow-up. Geometric perfusion deficits and VD in the MCP and DCP correlated with clinically significant outcomes at 1 year (P < 0.001). Eyes with these outcomes had lower VD and higher GPD, indicating worse nonperfusion of the deeper retinal layers than those that remained free from complication. These differences remained significant (P = 0.046 to < 0.001) when OCTA parameters were incorporated into models that also considered sex, baseline corrected visual acuity, and baseline DR severity. Adjusted receiver operating characteristic curve for DCP GPD achieved an area under the curve (AUC) of 0.929, with sensitivity of 89% and specificity of 98%. In a separate analysis focusing on high-risk proliferative diabetic retinopathy outcomes, MCP and DCP GPD and VD remained significantly predictive with comparable AUC and sensitivities to the pooled analysis. CONCLUSIONS: Evidence of deep capillary nonperfusion at baseline in eyes with clinically referable NPDR can predict short-term DR complications with high accuracy, suggesting that deep retinal ischemia has an important pathophysiologic role in DR progression. Our results suggest that OCTA may provide additional prognostic benefit to clinical DR staging in eyes with high risk.

Macrophage-like Cells Are Increased in Patients with Vision-Threatening Diabetic Retinopathy and Correlate with Macular Edema.

Macrophage-like cells (MLCs) are potential inflammatory biomarkers. We previously showed that MLCs are increased in proliferative diabetic retinopathy (PDR) eyes. Vision-threatening diabetic retinopathy (VTDR) includes PDR, severe non-PDR (NPDR), and diabetic macular edema (DME). No prior data exist on MLCs in eyes with severe NPDR or DME. This prospective, cross-sectional optical coherence tomography-angiography (OCT-A) imaging study included 40 eyes of 37 participants who had NPDR classified as non-VTDR (n = 18) or VTDR (n = 22). Repeated OCT-A images were registered, averaged, and used to quantify the main outcome measures: MLC density and percent area. MLC density and percent area were correlated with clinical characteristics, NPDR stage, presence of DME, and OCT central subfield thickness (CST). In VTDR eyes, MLC density (2.6-fold, p < 0.001) and MLC percent area (2.5-fold, p < 0.01) were increased compared with non-VTDR eyes. Multiple linear regression analysis between MLC metrics and clinical characteristics found that MLC density was positively correlated with worse NPDR severity (p = 0.023) and higher CST values (p = 0.010), while MLC percent area was only positively associated with increased CST values (p = 0.006). MLCs are increased in patients with VTDR. Macular edema is the most strongly associated factor with increased MLC numbers in NPDR eyes.

Hemodynamic Effects of Anti-Vascular Endothelial Growth Factor Injections on Optical Coherence Tomography Angiography in Diabetic Macular Edema Eyes.

Purpose: To evaluate retinal hemodynamic responses to anti-vascular endothelial growth factor (VEGF) injection in eyes with diabetic macular edema using optical coherence tomography angiography (OCTA). We performed a comparison of two different thresholding methods to identify the most accurate for studying the vessel density (VD) in diabetic macular edema eyes. Methods: The study prospectively included 26 eyes of 22 subjects (aged 60.2 ± 13.7 years) who underwent OCTA scan before and after anti-VEGF injection (mean interval between OCTA = 31.1 ± 17.3 days). We analyzed adjusted flow index, VD, and Skeletonized vessel length density in the parafoveal area (3-mm annulus with a 1-mm inner circle), along with full-thickness fovea avascular zone area and central foveal thickness (CFT). Using averaged scans VD as the ground truth, we compared two different algorithms for VD at the different plexuses. Longitudinal changes were assessed using a generalized linear model correcting for central foveal thickness and Q-score. Results: We found significantly decreased adjusted flow index in the DCP layer (P = 0.010) at the follow-up. Furthermore, foveal avascular zone (P < 0.001) and central foveal thickness (P = 0.003) showed significant decrease on follow-up compared with baseline. Comparing the thresholding algorithms showed that vessel length density-based thresholding was more accurate for quantifying the DCP VD. Conclusions: The adjusted flow index decreased significantly in the DCP layer on follow-up OCTA scan, suggesting vascular flow disruption and decreased deep retinal perfusion after anti-VEGF injection. Our results also highlight the fact that the choice of thresholding method is particularly critical for DCP quantification in eyes with diabetic macular edema. Translational Relevance: Findings confirmed impaired deep retinal capillary flow after anti-VEGF injection.

Macrophage-like cells are still detectable on the retinal surface after posterior vitreous detachment.

The identity of vitreoretinal interface macrophage-like cells (MLCs) remains unknown and potential candidates include retinal microglia, perivascular macrophages, monocyte-derived macrophages, and/or vitreal hyalocytes. Since hyalocytes are detectable on the posterior vitreous surface after vitreous extraction in animals, we imaged patients with and without posterior vitreous detachment (PVD) to determine if hyalocytes are the principal MLC component. We performed repeated foveal-centered 3 × 3 mm OCT-A images from 21 eyes (11 no PVD and 10 PVD eyes). Images were registered, segmented, and averaged. The OCT slab from 0 to 3 microns above the internal limiting membrane was used to detect MLCs. We calculated MLC density and distribution in relation to the superficial vascular plexus for 3 vascular regions-on vessels, perivascular, and non-vascular. MLC density was 1.8-fold greater in the PVD group compared to the no PVD group (P = 0.04). MLCs in eyes with PVD were increased 1.9-fold on-vessel (P = 0.07), 1.9-fold in the perivascular region (P = 0.12), and 2.2-fold in non-vascular areas (P = 0.22). MLC density was not severely reduced after PVD, suggesting that the majority of MLCs are not vitreal hyalocytes. PVD status is an important parameter in future MLC studies.

Deep Capillary Geometric Perfusion Deficits on OCT Angiography Detect Clinically Referable Eyes with Diabetic Retinopathy.

PURPOSE: To evaluate the sensitivity (SN) and specificity (SP) of OCT angiography (OCTA) parameters for detecting clinically referable eyes with diabetic retinopathy (DR) in a cohort of patients with diabetes mellitus (DM). DESIGN: Retrospective, cross-sectional study. SUBJECTS: Patients with DM with various levels of DR. METHODS: We measured vessel density, vessel length density (VLD), and geometric perfusion deficits (GPDs) in the full retina, superficial capillary plexus (SCP), and deep capillary plexus (DCP) on 3 × 3-mm OCTA images. Geometric perfusion deficit was recently described as retinal tissue located further than 30 µm from blood vessels, excluding the foveal avascular zone (FAZ). We modified the GPD metric by including the FAZ as an additional variable. Clinically referable eyes were defined as moderate nonproliferative DR (NPDR) or worse retinopathy, or diabetic macular edema (DME). One eye from each patient was selected for the analysis based on image quality. We used a binary logistic regression model to adjust for covariates. MAIN OUTCOME MEASURES: Sensitivity, SP, and area under the curve (AUC). RESULTS: Seventy-one of 150 included eyes from 150 patients (52 with DM without DR, 27 with mild NPDR, 16 with moderate NPDR, 10 with severe NPDR, 30 with proliferative DR, and 15 with DME) had clinically referable DR. Geometric perfusion deficit metric that included the FAZ performed better than GPD in detecting referable DR in the SCP (P = 0.025) but not the DCP or full retina (P > 0.05 for both). Deep capillary plexus GPD had the largest AUC for detecting clinically referable eyes (AUC = 0.965, SN = 97.2%, SP = 84.8%), which was significantly larger than the AUC for vessel density of any layer (P < 0.05 for all) but not DCP VLD (P = 0.166). The cutoff value of 2.5% for DCP GPD resulted in a highly sensitive test for detecting clinically referable eyes without adjusting for covariates (AUC = 0.955, SN = 97.2%, SP = 79.7%). CONCLUSIONS: Vascular parameters in OCTA, especially in the DCP, have the potential to identify eyes that warrant further evaluation. Geometric perfusion deficits may better distinguish these clinically referable eyes with DR than standard vessel density parameters.

Perspectives on diabetic retinopathy from advanced retinal vascular imaging.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and the most common cause of acquired vision loss in adults worldwide. DR is associated with long-term chronic hyperglycaemia and its detrimental effects on the neurovascular structure and function of the retina. Direct imaging of the retinal vasculature and staging of DR has been traditionally based on fundoscopy and fluorescein angiography, which provide only 2D views of the retina, and in the case of fluorescein angiography, requires an invasive dye injection. In contrast, advanced retinal imaging modalities like optical coherence tomography angiography (OCTA) and adaptive optics (AO) are non-invasive and provide depth-resolved, 3D visualization of retinal vessel structure as well as blood flow. Recent studies utilizing these imaging techniques have shown promise in evaluating quantitative vascular parameters that correlate tightly to clinical DR staging, elucidating functional changes in early diabetes, and monitoring DR treatment response. In this article, we discuss and synthesize the results of advanced retinal imaging studies in DR and their implications for our clinical and pathophysiologic understanding of the disease. Based on the recent literature, we also propose a model to describe the differential changes in vascular structure and flow that have been described on advanced retinal imaging as DR progresses. Future studies of these imaging modalities in larger and more diverse populations, as well as corroboration with histological and functional studies, will be important to further our understanding of DR.

Early-stage macular telangiectasia type 2 vascular abnormalities are associated with interdigitation zone disruption.

PURPOSE: To investigate the relationship between disruption in different photoreceptor layers and deep capillary plexus (DCP) telangiectasias in eyes with macular telangiectasia type 2 (MacTel). METHODS: 35 eyes (21 patients) with MacTel imaged with optical coherence tomography angiography (OCTA) were included. Circumscribed areas of DCP telangiectasia were traced from OCTA slabs and the corresponding spectral-domain OCT (SD-OCT) slabs were used to visualize the photoreceptor layer interdigitation zone (IZ) and ellipsoid zone (EZ). IZ attenuation, IZ loss, and EZ loss were graded by reviewing en face SD-OCT slabs for hypo-reflective areas and confirming their status on cross-sectional views. Total area of photoreceptor disruption and overlap with DCP telangiectasia were evaluated with respect to OCT-based MacTel stage. Longitudinal changes were evaluated in a subset of patients with follow-up imaging. RESULTS: Overlap of DCP telangiectasia with IZ attenuation significantly decreased with MacTel severity, while overlap with IZ and EZ loss significantly increased. Overlap with IZ loss peaked in moderate MacTel (Stages 3-5). Longitudinal imaging showed that new EZ loss at 6 months was largely predicted by baseline IZ loss. CONCLUSIONS: Worsening MacTel severity is characterized by greater overlap between DCP telangiectasia and zones of increasing severity of photoreceptor disruption, with EZ loss enlarging over time within areas of preexisting IZ disruption. We suggest that IZ disruption may indicate early photoreceptor dysfunction that eventually progresses to EZ loss, with IZ loss being a more reliable metric than IZ attenuation. Additional studies will be necessary to further explore long-term photoreceptor changes and evaluate their relationship with visual function in MacTel.

A Multivariable Predictive Model for Success of External Cephalic Version.

OBJECTIVE: To create a prediction model for external cephalic version (ECV) success using objective patient characteristics. METHODS: This retrospective study included pregnant individuals of at least 18 years of age with a nonanomalous, singleton gestation who underwent an ECV attempt between 2006 and 2016 at a single quaternary care hospital. Variables assessed included maternal age, height, weight, body mass index (BMI), parity, fetal sex, gestational age, estimated fetal weight, type of fetal malpresentation, placental location, and amniotic fluid volume. Univariable and multivariable logistic regression models were used to determine the association of patient characteristics with ECV success. Estimated odds ratios and corresponding 95% CIs were calculated for each variable, and backward elimination and bootstrapping were used to find a parsimonious model for ECV success with the highest discriminatory capacity (as determined by the area under the receiver operating characteristic curve [AUC]). This model was evaluated with a calibration curve across deciles of success. RESULTS: A total of 1,138 individuals underwent an ECV attempt and were included in this analysis. The overall ECV success frequency was 40.6%. Factors significantly associated with ECV success were maternal age, parity, placental location, estimated fetal weight, and type of fetal malpresentation. A final model with BMI, parity, placental location, and type of fetal malpresentation had the highest AUC (0.667 [95% CI 0.634-0.701]), resulted in good calibration, and is represented by the following equation: 1/[1+e-x] where x=1.1726-0.0314 (BMI)-0.9299 (nulliparity)+1.0218 (transverse or oblique presentation at ECV)-0.5113 (anterior placenta). An interactive version of this equation was created and can be accessed at www.ecvcalculator.com. CONCLUSION: A prediction model that estimates the probability of ECV success was created and internally validated. This model incorporates easily obtainable and objective patient factors known before ECV and may be used in decision making and patient counseling about ECV.

Macrophage-Like Cell Density Is Increased in Proliferative Diabetic Retinopathy Characterized by Optical Coherence Tomography Angiography.

Purpose: To quantitatively characterize macrophage-like cells (MLCs) at the vitreoretinal interface in different severity stages of diabetic retinopathy (DR) using optical coherence tomography angiography (OCTA). Methods: The study included 72 eyes of 72 subjects: 18 healthy controls, 22 diabetes mellitus (DM) without DR, 17 nonproliferative DR (NPDR), and 15 proliferative DR (PDR). We obtained repeated (average, 6.5; range, 3-10) macular OCTA scans for each eye. We registered and averaged the 3-µm OCT slab above the vitreoretinal interface to visualize MLCs. Using a semiautomated method, we binarized and quantified MLCs and compared MLC densities among groups. We also evaluated MLC distribution relative to underlying superficial capillary plexus vasculature and quantified MLCs overlying blood vessels within the perivascular 30-µm watershed region and within ischemic zones (defined as >30 µm from the nearest vessel). Results: MLC density was 2.8- to 3.8-fold higher in PDR compared with all other groups (P < 0.05 for all). MLC density in PDR was most increased in perivascular areas (3.3- to 4.2-fold; P < 0.05 vs. all) and on blood vessels (3.0- to 4.0-fold; P < 0.05 vs. all), and elevated to a lesser extent in ischemic areas (2.3- to 3.4-fold; P < 0.05 vs. all). MLCs were more likely to localize on blood vessels in DM without DR, NPDR, and PDR (P < 0.05 for all), but not healthy eyes. Conclusions: MLC density was significantly increased in PDR. MLCs clustered on blood vessels in diabetic but not in healthy eyes. Further studies are needed to confirm the origin, identity, and function of MLCs during DR.

Patient characteristics associated with complications of external cephalic version.

BACKGROUND: Patient characteristics associated with external cephalic version success are well documented; however, the association between patient characteristics and the likelihood of external cephalic version complications is poorly understood. OBJECTIVE: This study aimed to assess the frequency of patient characteristics associated with complications that lead to unanticipated delivery during external cephalic version. STUDY DESIGN: This retrospective study included pregnant women aged at least 18 years with singleton gestations who underwent an external cephalic version attempt between 2006 and 2016 at a single quaternary care center. External cephalic version complications were defined as persistent nonreassuring fetal status, placental abruption, labor, spontaneous rupture of membranes, and umbilical cord prolapse. Complications were only considered if they led to unanticipated induction or cesarean delivery within 24 hours of external cephalic version. Patient characteristics including maternal age, height, weight, body mass index, parity, fetal sex, gestational age, estimated fetal weight, the type of fetal malpresentation, and placental location were analyzed for their association with external cephalic version complications using univariable analysis and multivariable logistic regressions. RESULTS: Among the 1138 patients included in this analysis, external cephalic version complications occurred in 6.7% (n=76). These complications (not mutually exclusive) included nonreassuring fetal status (4.8%, n=55), placental abruption (1.6%, n=18), labor (1.0%, n=11), spontaneous rupture of membranes (0.6%, n=7), and cord prolapse (0.1%, n=1). Neonatal outcomes for those with complications included intensive care unit admission (10.5%, n=8), 5-minute Apgar scores <5 (1.3%, n=1), cord arterial pH <7 (6.6%, n=5), head cooling (1.3%, n=1), and anemia (6.6%, n=5). There were no perinatal deaths. In multivariable analysis, higher body mass index (adjusted odds ratio, 0.90 per kg/m2; 95% confidence interval, 0.84-0.97) and estimated fetal weight (adjusted odds ratio, 0.998 per gram; 95% confidence interval 0.998-0.999) were associated with decreased likelihood of experiencing external cephalic version complications, whereas greater gestational age at procedure (adjusted odds ratio, 1.95 per week; 95% confidence interval, 1.4-2.7) and anterior placental location (adjusted odds ratio, 2.0; 95% confidence interval, 1.1-3.7) were associated with increased likelihood of experiencing external cephalic version complications. CONCLUSION: In this large series, complications that led to delivery during external cephalic version occurred in 6.7% patients and were associated with body mass index, estimated fetal weight, gestational age, and placental location.

Exploring the Relationship Between Multilayered Choroidal Neovascularization and Choriocapillaris Flow Deficits in AMD.

Purpose: We used optical coherence tomography angiography to test the hypothesis that more complex, multilayered choroidal neovascular (CNV) membranes in AMD are associated with worse flow deficits (FD) in the choriocapillaris. Methods: Retrospective, cross-sectional study including 29 eyes of 29 subjects with neovascular AMD. En face choriocapillaris images were compensated for signal attenuation using the structural OCT slab and signal normalization based on a cohort of healthy subjects. We binarized the choriocapillaris using both local Phansalkar and global MinError(I) methods and quantified FD count, FD density, and mean FD size in the entire area outside the CNV, in the 200-µm annulus surrounding the CNV, and in the area outside the annulus. We used projection-resolved optical coherence tomography angiography to quantify CNV complexity, including highest CNV flow height, number of flow layers, and flow layer thickness. We explored the relationship between CNV complexity and choriocapillaris FD using Spearman correlations. Results: The highest CNV flow signal significantly correlated with lower FD count (P < 0.01), higher FD density (P < 0.05), and higher mean FD size (P < 0.05) in the area outside the annulus and the entire area outside the CNV using both Phansalkar and MinError(I). Within the annulus, CNV complexity was not consistently correlated with choriocapillaris defects. Conclusions: CNV vascular complexity is correlated with choriocapillaris FD outside the CNV area, providing evidence for the importance of choriocapillaris dysfunction in neovascular AMD, as well as the potential role of choroidal ischemia in the pathogenesis of complex CNV membranes.

Superficial capillary perfusion on optical coherence tomography angiography differentiates moderate and severe nonproliferative diabetic retinopathy.

PURPOSE: To identify objective optical coherence tomography angiography (OCTA) parameters that characterize the spectrum of non-proliferative diabetic retinopathy (NPDR), especially those that distinguish moderate from severe NPDR. METHODS: Sixty eyes of 60 patients with treatment-naïve NPDR (mild: 21, moderate: 21, severe: 18), 23 eyes with diabetes and no retinopathy, and 24 healthy control eyes were enrolled. OCTA slabs were segmented into superficial (SCP), middle (MCP), and deep capillary plexus (DCP) and thresholded by a new method based on DCP skeletonized vessel length. The foveal avascular zone (FAZ) area, parafoveal vessel density (VD), and adjusted flow index (AFI) from all three capillary layers and the vessel length density (VLD) of the SCP were compared between each severity group, after adjusting for age and image quality. RESULTS: All vessel density markers decreased with increasing severity of NPDR. SCP VD and VLD demonstrated significant differences between eyes with diabetes with no retinopathy and mild NPDR (p = 0.001 and p < 0.001, respectively), as well as between moderate vs. severe NPDR (p = 0.004 and p = 0.009, respectively). MCP VD significantly decreased between moderate and severe NPDR (p = 0.01). AFI significantly increased in the SCP and showed a decreasing trend in the MCP and DCP with increasing NPDR severity. CONCLUSIONS: Changes in the SCP VD, SCP VLD, and MCP VD can distinguish severe NPDR from lower-risk stages. SCP changes may be more reliable due to their lower susceptibility to noise and projection artifacts. Thresholding OCTA images based on DCP skeletonized vessel length showed less variability in moderate and severe NPDR. Additional studies are warranted to validate this new thresholding method.

Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.