Use of OCTA, FA, and Ultra-Widefield Imaging in Quantifying Retinal Ischemia: A Review

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1 REVIEW ARTICLe Use of OCTA, FA, and Ultra-Widefield Imaging in Quantifying Retinal Ischemia: A Review Chris Or, MD,* Almyr S. Sabrosa, MD,* Osama Sorour, MBChB, MSc,* Malvika Arya, BSc,* and Nadia Waheed, MD, MPH* Abstract: As ischemia remains a key prognostic factor in the management of various diseases including diabetic retinopathy, an increasing amount of research has been dedicated to its quantification as a potential biomarker. Advancements in the quantification of retinal ischemia have been made with the imaging modalities of fluorescein angiography (FA), ultra-widefield imaging (UWF), and optical coherence tomography angiography (OCTA), with each imaging modality offering certain benefits over the others. FA remains the gold standard in assessing the extent of ischemia. UWF imaging has allowed for the assessment of peripheral ischemia via FA. It is, however, OCTA that offers the best visualization of retinal vasculature with its noninvasive depth-resolved imaging and therefore has the potential to become a mainstay in the assessment of retinal ischemia. The primary purpose of this article is to review the use of FA, UWF, and OCTA to quantify retinal ischemia and the various methods described in the literature by which this is achieved. a unique opportunity to evaluate the peripheral retina, which is useful for the diagnosis, monitoring, treatment, and prognostication of various retinal pathologies. However, the emergence of 3- dimensional, noninvasive imaging by optical coherence tomography angiography (OCTA) has provided an opportunity to quantify vessel density in a defined retinal area and to gauge its loss over a period of time, either physiologically with aging or by underlying vascular pathology. 3,8,9 Recent studies have identified measurable parameters, such as vessel density, capillary length, intercapillary distance, and area of the foveal avascular zone (FAZ), to quantify the degree of retinal ischemia and to longitudinally assess its progression. 8,10 12 The primary purpose of this article is to review the use of FA, UWFA, and OCTA to quantify retinal ischemia and the various methods described in the literature by which this is achieved. Key Words: ultra-widefield retinal imaging, optical coherence tomography angiography, fluorescein angiography, ischemia, retina (Asia-Pac J Ophthalmol 2018;7:46 51) Inner retinal layers are richly perfused by the central retinal artery and its branches, whereas the choriocapillaris nourishes the photoreceptor layer. 1 Blood supply to the retina is susceptible to macro- and microvascular occlusion, where sudden occlusion of a major vessel may result in acute retinal ischemia with profound visual loss. On the other end of the spectrum, there is chronic microvascular occlusion, such as that encountered in diabetic retinopathy (DR), which is associated with alterations of retinal vasculature and eventual loss of capillary perfusion. 2,3 Prior studies have shown variation in the susceptibility of various retinal regions to ischemic insult, with predominant vulnerability to hypoperfusion in the midretinal periphery and the middle retinal layers. 4 7 Fluorescein angiography (FA) has been a gold standard in diagnosing and delineating the extent of retinal ischemia. Additionally, ultra-widefield fluorescein angiography (UWFA) offers From the *New England Eye Center, Tufts Medical Center, Boston, Massachusetts; and Institute Ophthalmology Rio de Janeiro/Hospital da Gamboa, Rio de Janeiro, Brazil. Received for publication January 15, 2018; accepted January 29, N.W. is a consultant for Optovue and provides research support for Nidek, Topcon, and Carl Zeiss Meditec. The authors have no other funding or conflicts of interest to declare. Reprints: Nadia Waheed, MD, MPH, New England Eye Center at Tufts Medical Center, 260 Tremont Street, Boston, MA E mail: nadiakwaheed@ gmail.com. Copyright 2018 by Asia Pacific Academy of Ophthalmology ISSN: DOI: /APO fluorescein angiography Fluorescein angiography remains the gold standard for detecting retinal vascular pathology since Novotny and Alvis first applied this technique in humans in However, its axial and lateral resolution, using traditional cameras and even the more recent scanning laser ophthalmoscopes, allow for limited visualization of capillaries and of the deep retinal vasculature. 13,14 Despite this, it has been used extensively in clinical trials and is currently the best characterized way of quantifying retinal ischemia. Although evaluation of retinal perfusion using FA is mainly qualitative, quantification of blood flow has been described using the principle of dye dilution tracking. In this method, the concentration of fluorescein dye within the blood at a specific observation point is graphed over time, producing a dye dilution curve. This principle was first employed by Hickam and Frayser 15 in 1965, when they published the first paper on quantifying retinal blood flow. They calculated the mean retinal circulation time [MCT; sometimes named mean transit time (MTT)] from a series of fluorescein photographs taken 1.5 seconds apart, which results in a preliminary estimation of retinal perfusion. 15 Hansen et al 16 used the following 3 parameters to quantify retinal perfusion in studying patients with ocular ischemic syndrome: arteriovenous passage time 1 as the interval between first appearance of fluorescein-stained blood in the temporal arcade arteries and its first appearance in the accompanying vein, arteriovenous passage time 2 as the interval between the first appearance in artery and the end of venular laminar phase, and venous filling time as the interval between first appearance in vein and the end of laminar phase. Similarly, Bertram et al 17 studied arm retinal circulation time, which is the time interval between injecting the dye in the antecubital vein and its first appearance in the retinal circulation, in diabetics and normal subjects and found no significant difference between both groups. Kim and co-workers 1 introduced a 46

2 Quantifying Retinal Ischemia semiautomatic protocol for generating a functional color-coded parametric image of retinal circulation, using parameters such as MTT. However, Tomic et al 18 concluded that the relationship between retinal blood flow and the measurement of transit times is a weak one, as accurately timed fluorescein angiograms are difficult to acquire and there are several variables that make interpretation difficult. Therefore, such methods are seldom used in clinical practice outside of academic institutions and only in situations that cause fairly significant changes in timed parameters, such as retinal artery occlusions. Macular ischemia (MI), defined as an enlargement of the FAZ and areas of perimacular capillary nonperfusion, is an important clinical feature in DR and other retinal vascular disorders, such as central retinal vein occlusion (CRVO). 8 Changes in the FAZ have been shown to be a good indicator of capillary dropout, which in turn are associated with disease progression. In particular, enlargement of the FAZ seems to be correlated with reduced visual acuity. 19,20 The FAZ outline was classified according to the Early Treatment Diabetic Retinopathy Study grading system as previously described. 21 This qualitative grading was found to be in good agreement with quantitative FAZ measurements. 22 Conrath et al 22 also noted that FAZ area was significantly increased in diabetics over nondiabetics and correlated with severity of DR. The same group was among the first to publish on a semiautomatic algorithm to detect and measure the FAZ. 23 Rasta et al 24 used a program that utilizes homomorphic filtering to correct light illumination and detect the areas of nonperfusion surrounded by healthy capillaries on FA images in patients with DR. This automated method had a sensitivity of 81% and an accuracy of 91% when compared with manual delineation performed by clinical experts. 25 Furthermore, Zheng et al 26 developed a software to allow for automated measurements of FAZ on FA photos, which demonstrated good reproducibility and a linear correlation with manual grading by retina specialists. Remky et al 27 demonstrated significant changes in the mean perifoveal intercapillary area, size of the FAZ, and mean capillary blood flow in patients with CRVO when compared with controls on FA. However, only mean capillary blood flow correlated with visual acuity in this study. Ultra-Widefield Imaging Since the introduction of human flash photography in 1886, significant advances have occurred in the diagnosis and management of retinal diseases. Traditional fundus cameras take images covering only degrees of the field of view, making it difficult to visualize pathology in the peripheral retina. In contrast, UWF imaging covers up to 200 degrees of the field of view, facilitating the acquisition of FA, indocyanine green angiography (ICG), pseudocolor and fundus autofluorescence (FAF). 28 Ultra-widefield imaging has been shown to be of use in the diagnosis and monitoring of various retinal diseases including retinal dystrophies, 29 DR, 30 and retinal vein occlusion (RVO). 31 Moreover, this technology allows better visualization of ischemic areas, vascular leakage, microvascular abnormalities, and neovascularizations (NV) in the peripheral retina. 32 Many early studies focused on DR and linked peripheral nonperfusion with NV, macular edema, and vascular leakage. 2,33 Fan et al 10 used manual segmentation of the nonperfused area (NPA) and total visible retinal area and discerned that in patients with treatmentnaive diabetic macular edema, the NPA in the midperiphery was negatively associated with central macular thickness. They also suggested that the global NPA was not associated with macular thickness or volume. 10 Silva et al 34 reported that increasing NPA was associated with worsening DR and the presence of predominantly peripheral lesions. However, they found that NPA was not associated with clinically significant macular edema or visual acuity. 34 The clinical utility of assessment of peripheral ischemia and predominantly peripheral lesions is being investigated as part of Diabetic Retinopathy Clinical Research Network clinical trials. Studies on CRVO further stressed the importance of the ischemic index (ISI), which is measured as the pixels contained in the NPA divided by the pixels in the whole retina surface based on UWFA. 35 In patients with RVO, the extent of retinal nonperfusion seen on UWFA is directly associated with the severity of macular edema, its resolution after treatment, 31 and the number of intravitreal ranibizumab injections required for treatment, emphasizing its importance in the diagnosis, follow-up, and treatment of these patients. 36 Singer et al 31 reported that patients with RVO and an ISI of greater than 10% had increased foveal thickness and worse visual acuity compared with those with an ISI of less than 10%. Kim et al 37 also reported that diabetic patients with peripheral nonperfusion, described as nonperfusion greater or equal to 5 disc areas in the periphery, and late peripheral vascular leakage had greater risk of recurring vitreous hemorrhage after vitrectomy. Mir et al 23 employed a qualitative approach to describe a reduction in retinal nonperfusion in patients with RVO undergoing ranibizumab injections. Querques et al 38 described ISI reduction in patients with DR after intravitreal dexamethasone implant, along with a reduction in central macular thickness. Additional measures of ischemia measured on UWFA include the peripheral leakage index, calculated as the area of capillary leakage expressed as a percentage of the total retinal area. Silva et al 34 reported a correlation between both the ISI and peripheral leakage index and the FAZ area in patients with DR, but no correlation between either was found with a reduced visual function. As the inner surface of the eye is nearly spherical, significant distortion is induced when the image is mapped onto a flat surface, with the distortion increasing with distance from the center. Additional limitations to the interpretation of UWF imaging include individualized contrast variations and the presence of artifacts. Due to this, many studies have utilized a categorical rather than absolute interpretation of nonperfusion in UWF imaging. 23 Furthermore, the peripheral areas can seem magnified when compared with the posterior pole, with the horizontal axis seemingly artificially stretched when compared with the vertical axis. Correction for this with a stereographic projection software algorithm, which corrects for the peripheral distortion and yields images that maintain the same angular relationship at every eccentricity, permits the determination of areas of nonperfusion in square millimeters rather than as a percentage. 25,39 This allows for better comparison of results from different studies. 39 However, it is uncertain how well the areas of nonperfusion correlate with the previously described ISI. 39 Furthermore, variations in image quality could lead to error in quantifying the area of ischemic retina in each image. Future studies must not only ascertain the significance of peripheral vascular pathology, but also standardize and automate the calculation of an ischemic index and outline accurately the areas of capillary dropout for it to be incorporated into clinical practice Asia-Pacific Academy of Ophthalmology 47

3 Or et al Optical Coherence Tomography Angiography As ischemia remains a key prognostic factor in the management of various diseases including DR, an increasing amount of research has been dedicated to its quantification as a potential biomarker. Earlier efforts using standard fundus photographs proved fruitful but laborious. 22,40 Fluorescein angiography is currently the primary method for evaluating retinal ischemia, but its invasive nature and the effects of variables including variable contrast, timing of dye injection, obscuration from leakage, degree of pigmentation, and media opacity make its use suboptimal, particularly in early detection of various diseases. 26 In clinical practice, the interpretation of retinal ischemia on FA remains semiquantitative and mostly qualitative. 8 Optical coherence tomography angiography is a noninvasive, dye-free OCT-based imaging technique that allows for volumetric visualization of vasculature. Multiple sequential OCT-B scans are acquired in rapid succession and then compared; the movement of blood cells from one image to the next creates a decorrelation signal that is then used to generate 3-dimensional angiograms of retinal and choroidal vasculature. Two of the major advantages of OCTA as compared with FA are its ability to visualize in great detail and high resolution the microvasculature of the eye and its 3-dimensional nature. This allows for the separation of the superficial from the deep capillary plexus and for detailed visualization of the deep plexus. Furthermore, Park et al 41 recently described the visualization of the middle capillary plexus, a vascular network separate from the superficial and deep capillary plexus, in healthy and diseased eyes. As the FAZ can be clearly delineated manually or via automated software algorithms, earlier studies in quantification of retinal ischemia focused on the FAZ, particularly in diseases such as DR. 26 Although FAZ area varies greatly in the population, 42 Takase et al 43 reported FAZ enlargement in DR subjects using en face OCTA images, a finding previously reported in studies using FA. 22,40,44,45 Furthermore, a study from our research group evaluating early microvascular changes in diabetic subjects without clinical retinopathy also supported these results, adding that qualitative features on OCTA, such as FAZ remodeling, microaneurysms, and venous beading, were more common in diabetics than in controls. 46 In contrast to previous FA studies that reported a significant increase in FAZ area with increasing severity of DR, 22 Takase et al 43 noted no statistical difference in FAZ area when comparing those with nonproliferative DR and proliferative DR. Other later and larger studies, however, have demonstrated significant increases in FAZ area with increasing DR severity. 47 Using swept-source OCTA (SS-OCTA), Al-Sheikh et al 48 reported an increase in mean FAZ area, which was measured using automatic detection software, and mean vessel density in the deep and superficial retinal layer in diabetics when compared with controls. Enlargement of the FAZ was also noted in other ischemic diseases such as RVO. 49,50 Assessing FAZ is a reliable and practical method of quantifying ischemia using OCTA. However, further studies are required to elucidate its clinical significance and reliability. Various studies have utilized vessel density measured on OCTA as a method of ischemia quantification. Agemy et al 51 reported a significant decrease in the skeletonized capillary density of all eyes with DR when compared with control eyes. Combining the skeletonized capillary density map and the vascular 48 perfusion map of each layer, Agemy et al 51 produced a capillary perfusion density map for each vascular layer of the retina. Using this technique, they reported a significant decrease in capillary perfusion density in all retinal capillary layers as DR progresses, with the most prominent changes noted in the deep capillary layer. 51 Other groups have investigated different indices of capillary density and morphology on OCTA, including vessel density and fractal dimension, and its association with disease. 52 Kim et al 52 reported an association between worsening DR and decreasing capillary density, decreasing branching complexity, and increasing vascular caliber. Kim et al 53 also demonstrated a significant difference in all vessel analysis metrics excluding vessel diameter index between the mild nonproliferative DR group and controls, which may make it useful in the early detection of DR. It should be noted that both vessel density, as the percentage of the image covered by vessels, and skeletonized vessel density, which some have argued to be a more sensitive measure of capillary perfusion, were investigated in this study. 52 Zahid et al 54 supported these results and reported a decrease in mean vessel density in the superficial and deep capillary layers in diabetics when compared with controls. However, this study did not find an association between vessel density or fractal dimension and DR severity, likely due to its small sample size. 54 Using manual segmentation, Salz et al 55 also evaluated the perifoveal intercapillary area, which increased with DR severity. Similar metrics were utilized in other ischemic diseases such as RVO. Studies have demonstrated a decrease in both the superficial and deep capillary plexus vessel density, fractal dimension, and skeletal vessel density being associated with severity of RVO. 50,56,57 Qualitative morphological findings such as perifoveal capillary arcade disruption and nonperfused grayish areas were noted to correlate with peripheral retinal capillary density in subjects with uveitis when compared with controls, using both semiautomated and manual segmentation methods. 53 In addition to identifying other vascular pathologies including microaneurysms and NV in DR, OCTA scans can also better delineate areas of retinal nonperfusion when compared with FA. 43 Takase et al 43 reported the area of nonperfusion in patients with DR to be greater in the superficial plexus than the deep plexus. Ishibazawa 58 also used OCTA to manually delineate nonperfused areas in the superficial and deep plexuses in DR subjects, revealing a difference between the extent of nonperfused areas between the plexuses. Using an automatic threshold-based approach, Jia et al 59 quantified areas of macular nonperfusion in DR, and Hwang et al 8 showed that the total nonperfused area is significantly higher in DR subjects when compared with controls. Similarly, Nesper et al 60 reported that OCTA parameters including FAZ area, vessel density, and percent area of nonperfusion demonstrated a linear correlation with DR severity. Adjusted flow index is the average signal strength in the region of interest after noise cancellation in the images and has been noted to decrease in the deep capillary plexus with increasing DR severity. 60 Flow index, which is the average decorrelation value in the region of interest of the en face retinal angiogram, gives information regarding blood vessel area and blood flow and has been investigated as a marker of ischemia. 61 Using an automated customized program on the superficial capillary plexus in patients with DR, Tang et al 11 investigated measures such as FAZ circularity index, which is the ratio of measured area of FAZ to the projected area of the circle, and vessel diameter index, which is calculated as the area occupied by blood vessels from the binarized image over the total length of 2018 Asia-Pacific Academy of Ophthalmology

4 Quantifying Retinal Ischemia blood vessels from the skeletonized image. They found that DR severity was associated with increased FAZ area, decreased FAZ circularity, lower vessel density, lower fractal dimension, and increased vessel diameter index, with a decrease in FAZ circularity being correlated with a reduction in visual function. 11 With the ability to correct for the eye s aberrations, adaptive optics offers much improved resolution of the vasculature in comparison with conventional FA. Mo and colleagues 62 used this technique to investigate skeletonized microvasculature parameters including FAZ area, perimeter, acircularity index, lumen diameter, and vessel density defined as total vessel length divided by the area and compared the results with those obtained via OCTA. The results were comparable between both modalities in FAZ metrics and vessel density, with an increased lumen diameter noted on OCTA over adaptive optics scanning laser ophthalmoscopy FA. 62 Swept-source OCTA has allowed researchers to better visualize the choriocapillaris. Using variable interscan time analysis, Moult et al 63 have demonstrated varying degrees of focal and diffuse choriocapillaris flow impairment under regions of geographic atrophy. Further studies have utilized similar techniques to visualize flow impairment and ischemia in diseases such as DR. 64 In addition, an increasing amount of research has been dedicated to automatic algorithms for the calculation of various ischemic markers. Using a prototypical SS-OCTA machine, Schottenhamml et al 65 evaluated a fully automatic algorithm to quantify capillary dropout, or intercapillary areas, in DR and reported a trend of increased intercapillary area with DR severity. Noting that most reported studies focused on either 3 3 mm or 6 6 mm fields of view centered on the macula, our research group has also investigated the use of a semiautomatic, texturebased algorithm to detect capillary nonperfusion and applied it to mm OCTA scans centered on the macula. A general trend of increasing capillary nonperfusion with increasing DR severity was observed (unpublished data). Several pitfalls exist in current OCTA studies that may confound the interpretation of the data. Segmentation methods and metrics calculations were not standardized and may not be comparable between studies, making it difficult to compare across studies. The presence of artifacts also results in a large percentage of data to be excluded. 66 Projection artifact also likely artificially increased the number of findings in the deep retinal layer in a number of studies. Presence of retinal abnormalities and pathology such as macular edema could also lead to retinal distortion, which further complicates data interpretation. However, softwareand hardware-based approaches that allow for a reduction of motion and projection artifacts may allow for more robust evaluation of these parameters in the future. Quantitative parameters on OCTA have the potential to provide novel objective markers for eye diseases, which may facilitate diagnosis, monitoring, and treatment. However, further clinical trials are needed for validation and reliability assessment of these metrics and to evaluate their utility in prognostication for them to be accepted and possibly used in the clinical setting. Conclusions Overall, both qualitative and quantitative assessment are essential in evaluating and monitoring ischemia. Advancements in both aspects have been made with the imaging modalities of FA, UWF, and OCTA, with each imaging modality offering certain benefits over the others. Fluorescein angiography remains the gold standard in assessing the extent of ischemia. Ultra-widefield imaging has allowed for the assessment of peripheral ischemia via FA. It is, however, OCTA that offers the best visualization of retinal vasculature 67 with its noninvasive depth-resolved imaging and therefore has the potential to become a mainstay in the assessment of retinal ischemia. However, a major limitation of OCTA is its limited field of view, which is a strength of UWFA imaging. Commonly employed macular scan patterns are 3 3 mm and 6 6 mm due to their higher resolution, which decreases as larger scan patterns are employed. Swept-source OCTA devices, with faster acquisition speeds compared with spectral-domain OCTA devices, allow for larger scan patterns of 15 9 and Further increasing image size would mean a trade off in scan quality, with lower resolution and likely increased motion artifact due to longer acquisition times. However, a field of view comparable to that of FA can be constructed by montaging OCTA scans. This technique allows for the visualization and assessment of peripheral ischemia as well. However, quantification of these montages is an area that is still being explored. Manufacturers of OCTA devices recognize the utility of wide-field images and are in the early stages of employing proprietary montaging software. With advancements in OCTA of faster scanning speeds, wider field of view, projection artifact removal algorithms, motion correction techniques, 71 and quantification of flow metrics, OCTA may become an essential tool in the assessment of ischemia. 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