950513

research-article20202020
OED0010.1177/2515841420950513Therapeutic Advances in OphthalmologyA Markan, A Agarwal

Therapeutic Advances in Ophthalmology Review

Novel imaging biomarkers in diabetic

Ther Adv Ophthalmol

2020, Vol. 12: 1–16

retinopathy and diabetic macular edema DOI: 10.1177/

https://doi.org/10.1177/2515841420950513
2515841420950513
https://doi.org/10.1177/2515841420950513

© The Author(s), 2020.

Article reuse guidelines:
Ashish Markan*, Aniruddha Agarwal*, Atul Arora, Krinjeela Bazgain, Vipin Rana sagepub.com/journals-
permissions
and Vishali Gupta

Abstract: Diabetic retinopathy is one of the major microvascular complications of diabetes

mellitus. The most common causes of vision loss in diabetic retinopathy are diabetic macular
edema and proliferative diabetic retinopathy. Recent developments in ocular imaging have
played a significant role in early diagnosis and management of these complications. Color
fundus photography is an imaging modality, which is helpful for screening patients with
diabetic eye disease and monitoring its progression as well as response to treatment. Fundus
fluorescein angiography (FFA) is a dye-based invasive test to detect subtle neovascularization,
look for areas of capillary non-perfusion, diagnose macular ischemia, and differentiate
between focal and diffuse capillary bed leak in cases of macular edema. Recent advances
in retinal imaging like the introduction of spectral-domain and swept source-based optical
coherence tomography (OCT), fundus autofluorescence (FAF), OCT angiography, and ultrawide
field imaging and FFA have helped clinicians in the detection of certain biomarkers that can
identify disease at an early stage and predict response to treatment in diabetic macular
edema. This article will summarize the role of different imaging biomarkers in characterizing
diabetic retinopathy and their potential contribution in its management.

Keywords: biomarkers, diabetic macular edema, fluorescein angiography, optical coherence

tomography, optical coherence tomography angiography, retinopathy

Received: 10 April 2020; revised manuscript accepted: 13 July 2020.

Introduction tests are invasive, and their clinical applicability is Correspondence to:
Vishali Gupta
Diabetic retinopathy (DR) is one of the leading still under investigation. Imaging biomarkers pro- Professor of
causes of blindness in the middle-age popula- vide an in vivo assessment of the health of the Ophthalmology, Advanced
Eye Center, Post Graduate
tion.1 Among patients with DR, diabetic macular retina and choroid non-invasively. These tools Institute of Medical
edema (DME) is the leading cause of moderate provide a near-histological assessment of various Education and Research
(PGIMER), Sector 12,
visual loss.2 In the current era, imaging modalities alterations, including retinal capillary densities, Chandigarh 160012, India
play an important role in deciding the treatment non-perfusion, vascular remodeling, and foveal vishalisara@yahoo.co.in
protocol as well as prognosticating outcome. avascular zone (FAZ) area. Recently, there has Ashish Markan
Aniruddha Agarwal
They also have significant utility in screening been an interest in determining the prognostic Atul Arora
patients with diabetes mellitus to rule out any fea- value of changes on optical coherence tomogra- Krinjeela Bazgain
Vipin Rana
tures of DR at an early stage. phy (OCT) such as foveal intraretinal cysts and Advanced Eye Center,
edema, including size and location, presence of Department of
Ophthalmology, Post
Recently published literature on DME has subretinal fluid (SRF), and integrity of retinal lay- Graduate Institute of
focused on several novel clinical, laboratory, and ers in DME.4 Medical Education and
Research (PGIMER),
imaging biomarkers. Laboratory biomarkers that Chandigarh, India
aid in determining the disease severity and In this review, we have summarized various stud- *The two authors have
response to therapy include levels of cytokines ies that have provided insights into the utility of contributed equally to the
manuscript and share the
and inflammatory markers in serum, vitreous, tools such as OCT and OCT angiography first authorship.
aqueous, and tear fluid.3 However, most of these (OCTA) in DR and DME. This review provides

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Therapeutic Advances in Ophthalmology 12

on OCT imaging in the treatment of DME with

anti-VEGF agents. As OCT is non-invasive, it was
quickly adopted by clinicians for the assessment of
patients with DME. Currently, OCT is an invalu-
able and indispensable tool in patients with diabe-
tes to determine the need for treatment and
prognosticate patients with DME.

Disorganization of retinal inner layers

Disorganization of retinal inner layers (DRIL) is
defined as the inability to distinguish between the
ganglion cell layer–inner plexiform layer complex,
inner nuclear layer, and outer plexiform layer.
DRIL can be associated with or without center-
Figure 1. The optical coherence tomography (OCT) signs of diabetic involving DME.6 DRIL is measured on OCT
macular edema (DME). In panel (a), a patient with DME and multiple B-scans by looking at the central 1 mm retinal
hyperreflective retinal foci (HRF) is shown. The magnified image shows the zone. Disorganization of more than 50% or >500 µm
presence of disorganization of retinal inner layers (DRIL; yellow bracket) of this area is considered significant and is associ-
with multiple HRF mainly in outer retinal layers (yellow arrowheads). OCT ated with worse visual prognosis in eyes with
of another subject (b) shows the presence of few HRF in outer nuclear edema or resolved edema. DRIL has been found
layer with few cystic spaces (white arrow). The OCT scan of another
to be a reliable biomarker for prognosis of visual
patient shows bridging retinal processes between the cystic cavities (white
arrowheads) (c). acuity in DME. Inner retinal layers consist of
axons, bipolar cells, and nuclei of amacrine cells,
all of which are important for transmission of vis-
ual signals from photoreceptors to the ganglion
a comprehensive summary of various imaging cell layer. DRIL signifies damage to these struc-
techniques and novel biomarkers that help in tures leading to abnormal visual processing. Sun
determining the functional outcomes of subjects and colleagues found that early change in DRIL
with DR and DME. predicted the visual outcomes during the course
of treatment. An increase in DRIL during
Since this is a narrative review, no formal search 4 months predicted a decline of visual acuity by
strategy was used to compile the studies from one line.6 Figure 1 depicts DRIL.
literature.
Recent studies have correlated DRIL with the
amount of maculopathy present even in the absence
OCT of any DME.7 DRIL has been correlated with macu-
OCT is an imaging modality that provides high- lar capillary non-perfusion and size of FAZ.8,9 DRIL,
resolution cross-sectional images of the neurosen- in recent studies, has been associated with increasing
sory retina and choroid by processing the severity of DR, especially proliferative DR (PDR).10
backscattered light. With the advancement in tech- The presence of DRIL is associated with outer reti-
nology, better OCT tools are now available com- nal layer disruption, specifically ellipsoid zone (EZ)
pared with the previously available time-domain and external limiting membrane (ELM).10 Taken
OCT. Presently, spectral-domain (SD) and swept- together, DRIL appears to be a useful OCT bio-
source (SS) OCT are the most advanced technolo- marker for visual acuity assessment, capillary perfu-
gies available to image the fundus. The value of sion, and other morphological changes in DME.
OCT in DME was first established by the
Prospective OCT Study With Lucentis for Joltikov and colleagues11 have studied DRIL in
Neovascular AMD study, which used OCT as a diabetic patients with no evidence of DR or early
tool to monitor the intraretinal and SRF fluid to DR to elucidate the effects of DRIL in early neu-
decide further therapy with anti-vascular endothe- roretinal disease. It can be used as a potential
lial growth factor (anti-vascular endothelial growth clinical marker of visual function to test potential
factor [VEGF]) agents.5 Since then, various studies therapies for diabetic patients with early stages of
have used the pro re nata (PRN) approach based neuroretinal impairment.

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 A Markan, A Agarwal et al.

Hyperreflective retinal foci subjects with DME. Elevated VEGF level in DR

Hyperreflective retinal foci (HRF) appears as affects the inner blood–retinal barrier leading to
intraretinal hyperreflective dots on OCT in sub- increased vascular permeability resulting in a
jects with retinal pathologies such as DME decreased osmotic gradient, extracellular fluid
(Figure 1). HRF represent subclinical lipopro- accumulation, and cyst formation, while the outer
teins that extravasate after breakdown of inner blood–retinal barrier is not affected significantly
blood–retinal barrier.12,13 Subretinal HRF are (Figure 1).20,21 Cystoid spaces in DME, unlike
associated with subfoveal hard exudates after res- the cyst in cystoid macular edema (CME), can
olution of subretinal serous detachment.14 In cause photoreceptor damage and affect visual
contrast to other studies, Lee and colleagues15 outcomes.22 Large cysts (>200 µm) in the outer
proposed that HRF represent activated microglial nuclear layer (ONL) are seen in late stage of
cells since they determined the presence of DME and have a negative impact on macular
increased soluble CD14 in the aqueous humor. function (seen by microperimetry) than smaller
Soluble CD14 is released by activated microglial cysts or cystoid formation occurring in inner reti-
cells. HRF are initially present in the inner retinal nal layers.23,24 This is because cysts in the ONL
layers and subsequently migrate to the outer reti- have shown to damage photoreceptor cells and
nal layers. The features of HRF include size less disrupt inner segment–outer segment (IS/OS)
than 30 µm, absence of back-shadowing, and junction, thus causing irreversible damage to vis-
reflectivity similar to retinal nerve fiber layer. ual functions.25

HRF are important imaging markers of retinal Treatment with anti-VEGF therapy leads to a
inflammation.16 The size and number of HRF decrease in the number and size of ONL cysts by
may decrease after treatment with anti-VEGF decreasing the permeability of the inner blood–
and corticosteroid implants. In a recent study, retinal barrier. This was associated with improve-
corticosteroid implants were shown to have better ment in best-corrected visual acuity and
outcomes compared with anti-VEGF agents in microperimetric retinal sensitivity.24
subjects with DME in the presence of HRF. The
study showed that the initial presence of a larger Hyperreflective signals within the cyst are associ-
number of HRF responded better to dexametha- ated with severe disruption of the blood–retinal
sone implants than anti-VEGF therapy.17 Further barrier. There is paucity of information in the lit-
investigations that correlate HRF with visual acu- erature on the contents of these intraretinal
ity are needed. Recent literature suggests that a hyperreflective signals. In one study,26 the authors
higher number of HRF on SD-OCT is associated hypothesize that fibrin and other inflammatory
with early recurrence of DME after steroid by-products may fill these spaces. Treatment with
implant. As a result, patients with an increased anti-VEGF agents did not seem to directly change
number of HRF on OCT should be frequently their natural course.
followed up for early intervention if required.18
Kashani and colleagues27 have described this
intraretinal reflective material as suspended scat-
Hyperreflective choroidal foci tering particles in motion (SSPiM) on OCTA.
Hyperreflective choroidal foci (HCF) is a recently SSPiM which appears as an extravascular signal
described entity, which are HRF seen in choroidal on OCTA is likely due to Brownian motion of
layers in DME.19 Such HRF in choroid have been particles within intraretinal fluid pockets. SSPiM
described in Stargardt’s disease as lipofuscin depo- are usually found at vascular–avascular junctions
sitions. It is believed that HCF have migrated from and in some cases resolve with formation of hard
the retina into choroidal layers with disruption of exudates. Extravasated lipid is believed to be
the ELM and EZ. The presence of HCF is a poor involved in the formation of these SSPiM and
prognostic marker in terms of visual acuity. HCF subsequently hard exudates.
were found to be present significantly more in PDR
eyes than NPDR (non-proliferative DR) eyes. The intraretinal cystoid spaces can be categorized
based on their size. The cysts have been classified
as small (<100 µm), large (101–200 µm), or giant
Intraretinal cystoid spaces (>200 µm). Large cysts are associated with poor
The size and location of intraretinal cystoid spaces visual prognosis. Size of the cyst is correlated with
are relevant in the functional outcomes of the extent of macular ischemia. Both horizontal

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Therapeutic Advances in Ophthalmology 12

and vertical diameter of cyst increases with sever- of DME, extent of retinal involvement, and dif-
ity of macular ischemia.28 ferential involvement of inner versus outer retinal
tissue. Cross-sectional area of retinal tissue
Other parameters to be considered while analyz- between the plexiform layers in CME is a better
ing CME include location of the cyst about the predictor of visual acuity than macular thickness
center and its lateral extension, the degree of the (80% versus 14%).31
anatomical damage caused to the inner and outer
layers by the cystoid change, any associated pho-
toreceptor damage or retinal pigment epithelial Subfoveal choroidal thickness
(RPE) damage, and the presence of associated Studies suggest that baseline subfoveal choroidal
SRF.23,29 All these characteristics may influence thickness is a predictor of response to anti-VEGF
baseline visual acuity and visual outcomes in therapy.32 A greater baseline choroidal thickness
response to treatment. was independently associated with better ana-
tomical and functional response to anti-VEGF
therapy. Patients with greater choroidal thickness
Bridging retinal processes are presumed to have an intact choriocapillaris
Recently, a study by Farhan and colleagues30 and thus a less ischemic outer retina. This means
demonstrated that bridging retinal processes the photoreceptor function is well preserved in
which are seen in between the cystic cavities is these eyes, thus explaining superior visual out-
associated with improved visual acuity after treat- comes following anti-VEGF therapy.
ment. These tissues represent residual neural ele-
ments, which connect outer and inner retina and Choroidal thickness increases with increasing
thus help in transmitting visual impulses from the severity of DR, owing to increased VEGF pro-
inner retinal layers to the optic nerve axons. duction. Increased VEGF levels might result in
Although exact nature of these tissues is not vasodilation of choroidal vessels and subsequently
known, it is believed that Muller cells and bipolar increased choroidal thickness.33
cells are an important component.

In contrast to this, absence of these bridging reti- Photoreceptor outer segment

nal tissues between inner and outer retina have Photoreceptor outer segment (PROS) is defined
poor prognosis post treatment. These eyes are as the length between the photoreceptor inner
unlikely to improve despite resolution of cysts and outer segment junction and the RPE. Studies
and end up with foveal atrophy and thinning.31 have shown that PROS length correlated better
than macular thickness at the fovea with visual
acuity in patients with DME.34 Opsin plays an
Retinal thickness important role in regulation of visual cycle. It is
The primary cause for increased retinal thickness believed that outer segments which act as opsin
in DME is intra- and subretinal edema. Edema reservoirs are more important than inner seg-
occurs as a result of breakdown of blood–retinal ments as the source of adenosine triphosphate.
barrier and extravasation of lipid in the intrareti- Therefore, PROS may be considered as an impor-
nal space. Central subfield thickness (CST) can tant prognostic biomarker in eyes with DME.
be easily measured on SD-OCT, and measure-
ment of CST is invaluable in assessing the degree Ozkaya and colleagues35 have shown that PROS
of DME. When the retinal thickness increases length was significantly less in eyes with DR or
beyond the stretching capability limit of retina, it DME compared with healthy subjects or diabetic
can lead to damage to the bipolar axons leading to patients with no retinopathy. More extensive
decreased visual signal transmission. Thus, studies are required to judge the true predictive
despite resolution of DME, the gain in vision may power of this index in future.
be suboptimal. Often, resolution of DME is
accompanied by macular atrophy due to perma-
nent damage to the photoreceptors. An important Hard exudates
consideration is that CST is not a reliable indica- Hyperreflective spots which are larger than 30
tor of visual acuity. CST is not prognostic or pre- µm, associated with back-shadowing, present in
dictive of final outcomes. It is always important to the outer retinal layers, having reflectivity similar
look for other associated biomarkers like pattern to RPE–Bruch’s complex are suggestive of hard

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 A Markan, A Agarwal et al.

was associated with better visual gains at the end

of 1 year. These studies also showed a positive
impact from SRF in response to ranibizumab
therapy. Furthermore, eyes with SRF were
shown to have better visual gains in a study eval-
uating the effect of vitrectomy in diffuse DME.46

Post hoc analysis of VIVID-DME and VISTA-

Figure 2. Optical coherence tomography (OCT) scan DME studies showed that visual outcomes were
of a patient with diabetic macular edema shows a better with intravitreal aflibercept than laser,
thick posterior hyaloid membrane (TPHM; yellow regardless of baseline SRF status, although
arrowheads) with increased retinal thickening. There greater treatment effect of intravitreal aflibercept
are hard exudates in the inner retina (yellow asterisk) was seen in patients with baseline SRF than those
causing back-shadowing of OCT signal. who did not.47

Moon and colleagues48 have shown DME eyes

exudates in retinal OCT (Figure 2).36
Hard exu- with SRF to respond significantly with dexameth-
dates are lipo-proteinaceous (albumin and fibrin) asone implants and advocate the use of dexa-
deposits that accumulate due to breakdown of methasone implants in patients with DME with
inner blood–retinal barrier. Quantitative meas- SRF. Eyes with SRF have shown increased
urement of hard exudates in patients with DME Interleukin (IL)-6 levels, signifying active inflam-
has been associated with serum lipid levels; high mation in these eyes.49 Zun and colleagues4 have
triglyceride levels can increase the risk of central shown that the presence of SRF is associated with
macular involvement causing accumulation of a better response with dexamethasone implants.
subfoveal hard exudates.37,38 Quantitative infor-
mation may be useful to monitor progression of To conclude, our understanding of the relation-
hard exudates and treatment response in diabetic ship between SRF status and visual outcomes is
maculopathy.39 Intravitreal steroids (triamci- still evolving and will need further long-term
nolone, dexamethasone implants) may be more studies.
effective in reducing hard exudates in patients
with DME compared with anti-VEGF agents,
especially when the exudates are subfoveal.40 Integrity of ELM and EZ
The integrity of outer retinal layers is a direct
indicator of the health of the retinal photorecep-
Subfoveal neurosensory detachment tors and RPE. Studies have shown that eyes with
The prevalence of subfoveal serous retinal detach- intact IS/OS junction have better visual gains post
ment in subjects with DME is 15–30%. Serum treatment. IS/OS junction can be graded as com-
albumin has been shown to be a sensitive marker pletely continuous, partly disrupted and com-
for the presence of SRF.41 Hypoalbuminemia can pletely disrupted.4 Subjects with long-standing
lower the intravascular osmotic pressure. This DME may demonstrate loss of ELM and EZ
along with increased hydrostatic pressure can focally or in a diffuse manner. Studies have
cause retention of fluid in the subretinal space. revealed suboptimal visual acuity gains in patients
with outer retinal disruption. Visual acuity has
Presence of SRF in DME is an important OCT shown a positive correlation with the survival rate
biomarker. The role of SRF in final visual and of ELM and the EZ.14
anatomical outcomes is still confusing. There
are a number of studies which have shown that
the presence of SRF is associated with good ana- Taut posterior hyaloid membrane
tomical and functional gains. Contrary to this, Patients with DR demonstrate several abnormali-
there are other studies which have shown that ties in the vitreous referred to as diabetic vitreopa-
the presence of SRF is associated with poor vis- thy. The alterations in the vitreous result in
ual gains.42,43 Results from RESTORE study44 abnormal posterior vitreous detachment (PVD;
and post hoc analysis from RISE/RIDE study anomalous PVD) that is characterized by strong
proved the protective role of SRF.45 These stud- focal/diffuse retinal adhesions and incomplete
ies showed that the presence of baseline SRF detachment. Often, the posterior hyaloid forms a

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Therapeutic Advances in Ophthalmology 12

sheet along the posterior pole resulting in trac- 100 degrees are termed as ultrawide field imag-
tional force and mechanical retinal distortion. ing.58 With peripheral sweeps, it is possible to
This is termed as taut posterior hyaloid mem- obtain fundus images of a reasonable quality of an
brane (TPHM; Figure 2). TPHM is responsible additional 20–30 degrees. Since the earliest signs
for recalcitrant macular edema. Patients with of DR are seen in the mid-periphery, an extended
TPHM benefit by pars plana vitrectomy and field of view is crucial and desirable in the screen-
removal of the taut hyaloid.50,51 ing, diagnosis, and treatment of DR. Currently,
the ultrawide field imaging tools, which are cSLO
(confocal scanning laser ophthalmoscope) based,
Choroidal vascularity index provide a panoramic imaging of 82% of the retina
Choroidal vascularity index (CVI) was initially (200-degree) in a single frame with no need of
described by Agrawal and colleagues52 as a ratio mydriasis or contact lens.
of choroidal luminal area to total choroidal area.
CVI, unlike choroidal thickness, is unaltered by A number of studies have shown the utility of
ocular or systemic factors. CVI has been recently ultrawide field imaging in DR.59–61 Silva and col-
described as a novel OCT parameter to monitor leagues59 showed that additional peripheral reti-
the progression of DR.53 Studies have shown a nal findings were detected in ultrawide field
decrease in CVI with unaltered choroidal thick- images compared with the Early Treatment
ness in patients of DR. CVI has also been shown Diabetic Retinopathy Study (ETDRS) standard
to correlate with the progression of DR. Patients 7-field fundus photography. This resulted in bet-
with PDR have a significantly lower CVI than ter assessment of DR and documentation of addi-
those with mild to moderate NPDR.54 CVI can tional findings resulting in change in therapeutic
be altered even before the onset of DR suggesting decision-making in 10% eyes. In addition, studies
that choroidal vasculature may be the site of pri- have also correlated DME to peripheral retinal
mary insult in diabetic eye disease. ischemia.60,61 Wessel and colleagues60 found a
direct correlation of peripheral retinal ischemia
with DME on OCT. Patients with peripheral reti-
Fundus fluorescein angiography nal ischemia had 3.75 times more odds of devel-
Fundus fluorescein angiography (FFA) has been oping DME as compared with the patients with
considered the gold standard in studying the reti- no peripheral retinal ischemia. It is believed that
nal vasculature.5 FFA can help in determining this retinal ischemia is responsible for increased
various changes such as retinal capillary non-­ VEGF production. Patients with extensive
perfusion, vascular telangiectasia, capillary drop- peripheral ischemia should be followed up more
outs, enlargement or irregularity of the FAZ, and closely than with no retinal ischemia. These
the presence of neovascularization. Since FFA patients can respond better with targeted retinal
allows dye leakage and pooling, it allows rapid photocoagulation in adjunct to anti-VEGF and
assessment of retinal vascular changes, some of macular laser therapy. Studies have shown that
which may be missed on routine dilated fundus mean decrease in central macular thickness is cor-
examination (for instance, occult retinal neovas- related inversely with ischemic index measured
cularization). FFA is an important tool to differ- on widefield imaging. Ongoing Protocol AA aims
entiate between intraretinal microvascular to compare the ultrawide field fundus imaging
anomalies (IRMAs) and neovascularization else- with ETDRS seven-standard-fields imaging for
where (NVE).5 FFA helps to differentiate between the assessment of DR and predict rates for wors-
focal leak and diffuse capillary bed leak in cases of ening of DR.
DME.55 Before the introduction of OCTA, FFA
was used to assess the health of the macula and
degree of macular ischemia.56 Biomarkers on FFA
The earliest signs of DR, microaneurysms, might
not be evident on clinical examination.62,63 They
Ultrawide field imaging and angiography are highlighted in FFA as punctate areas of hyper-
Conventional fundus imaging and angiography fluorescence (Figure 3).64 The dot and blot hem-
can provide valuable information that is limited to orrhages cause fluorescence block.65 Marked
the central 30–50 degrees of retina.57 Imaging aneurysm formation results in capillary closure,
angles more than 50 degrees are termed as wide- which is generally seen in mid-peripheral retina
field imaging, and imaging angles greater than that gradually increases toward the periphery.66–68

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 A Markan, A Agarwal et al.

maculopathy, there is a generalized breakdown of

the blood–retinal barrier that causes profuse early
leakage from the entire capillary bed of the poste-
rior pole.76

This is accompanied by cystoid changes in the

macula that can be appreciated in OCT. Ischemic
maculopathy is characterized by an enlargement
in the FAZ. CME is seen in a petaloid pattern
due to late staining of fovea with pooling of the
dye into parafoveal cyst-like spaces.70 FFA helps
in locating the leakage point in cases of macular
edema, therefore assisting in focal or grid laser for
treatment.77

Leakage pattern
Fluorescence leakage indicates breach in blood–
retinal barrier.78 Fluorescein, the fluorescent dye,
helps in tracking the leak. However, in cases of
neovascularization, the leakage obscures and
Figure 3. Ultrawide field fluorescein angiography blurs the image of a new vessel. Microaneurysms
(UWF FA) of a patient with proliferative diabetic
result in focal leakage, which cause focal macular
retinopathy (PDR) (a) shows a large leak from a nasal
retinal neovascularization (white arrowhead). There edema, whereas diffuse capillary bed leak due to
are peripheral capillary non-perfusion areas (yellow disruption of blood–retinal barrier causes diffuse
arrowhead). In the posterior pole, the FA shows the macular edema.
presence of macular capillary dropouts with vascular
telangiectasia (yellow arrows) and leakage. Another
patient with PDR on UWF FA (b) shows a large area of FAZ
capillary non-perfusion involving the posterior pole
FAZ is polygonal in normal eyes and it reflects
(yellow dashed square). This patient has been treated
with laser pan-retinal photocoagulation. microcapillary foveal circulation. FAZ is increased
in DR eyes which is the result of perifoveal capil-
lary occlusion.79 FAZ borders are seen to be irreg-
ular in DR eyes. However, FAZ dimensions do
Non-perfusion of capillaries leads to patchy areas not significantly correlate to fluorescence leakage,
of hypofluorescence (Figure 3).69 and it has not been correlated independently to
the staging of retinopathy.80,81
Macular ischemia is characterized by an increase
in FAZ.64 Dilated, tortuous, shunt vessels are seen
in the peripheral ischemic retina.70 Fluorescence OCTA
being 80% protein bound leaks out of incompe- OCTA is a novel exciting tool that provides
tent vessels suggesting a breach in blood–retinal detailed information of the retinochoroidal micro-
barrier.71 Leakage (Figure 3) is seen in retinal neo- vasculature. The information provided by OCTA
vascularization which is seen at the junction of supplements the information obtained using
perfused and non-perfused retina.64,72,73 Optic FFA, including precise areas of capillary non-­
disc neovascularization appears before retinal neo- perfusion, presence of collaterals or retinal/optic
vascularization suggesting a choroidal blood sup- nerve head neovascularization, and abnormalities
ply to the optic disc.70 of the FAZ.82 OCTA is advantageous and unique
because it can separately analyze each of the three
DME is a very common cause of decreased vision. retinal capillary plexi which is important for
Diabetic maculopathy has been grouped as focal, understanding the pathophysiologic changes in
diffuse, and ischemic maculopathy.74 In focal DR. OCTA produces static images of flow in the
maculopathy, focal leakage occurs from microa- retinal circulation as compared with FFA, which
neurysms with hard exudates arranged in a circi- produces dynamic images. OCTA images are
nate pattern around the leakage.75 In diffuse three-dimensional (3D) and can be

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Therapeutic Advances in Ophthalmology 12

Figure 4. Fundus photograph (50-degree view) of a patient with diabetic retinopathy shows a large
neovascular frond nasal to the optic disc (a). This is seen on the corresponding fluorescein angiography as
leakage from the neovascularization (b). Optical coherence tomography angiography shows the presence of
the large neovascularization (yellow arrowheads) (c).

manually segmented and co-registered with the morphological patterns of neovascularization at

accompanying structural OCT images. With the disc (NVD) have been described on OCTA which
advent of widefield OCTA and flow overlay, one can help us to detect the response to treatment.88
can differentiate between NVE and IRMA, both Figure 4 shows NVD and NVE detected on
of which signify an advanced stage of DR.17,83 OCTA.

OCTA can identify microaneurysms, IRMAs, Widefield OCTA has emerged as a promising
capillary non-perfusion area, and neovasculari- tool with a potential to replicate or replace FFA in
zation even before they are appreciated clinically diagnosing or monitoring the progression of the
or on fundus photography.84 Thompson and col- disease. Widefield OCTA has been compared
leagues85 detected microaneurysms on OCTA with ultrawide field FFA in patients with DR.
even before they were seen on dilated clinical Recent observational studies have shown wide-
examination. Although FFA has higher sensitiv- field OCTA to be comparable with ultrawide field
ity than OCTA on detecting microaneurysms, FFA in detecting NVs. It also allows to detect
studies have shown that OCTA can even detect subtle changes in NVs after treatment, thus pre-
microaneurysms, which are not picked up on dicting the future course of these lesions.89
FFA.85,86 Couturier et al. showed that the detection rate of
capillary non-perfusion areas was higher with
IRMAs on en face images appear as dilated or widefield OCTA than ultrawide field FFA.90 This
looping vessels near the areas of non-perfusion. is attributed to the fact that OCTA, unlike FFA,
OCTA has higher detection rates of IRMAs as is not affected by leakage and can clearly deline-
compared with color fundus photography. Other ate the abnormal vasculature and capillary non-
features which favor IRMAs include presence of perfusion areas. These studies suggest that
intraretinal hyperreflective dots and outpouching widefield OCTA has the potential to replace
of the internal limiting membrane (ILM). IRMA ultrawide field FFA in the future, in diagnosing
appears as focal areas of increased intraretinal and monitoring the changes in DR.
blood flow within the superficial capillary slab on
OCTA.87
Retinal vascular density
Standard OCT does not provide information Vessel density (VD) is defined as the ratio of
about IRMAs and their flow status. Structural blood vessel area to the total measured area.91 It
OCT B scan with a flow overlay will help in dif- decreases in both superficial capillary plexus
ferentiating between IRMA and NVEs. IRMA (SCP) and deep capillary plexus (DCP) in
will have flow signals within the retina under the patients with DR (Figure 5).92 VD has also been
ILM, whereas NVEs appear as flow voids above shown to decrease in diabetic patients without
the ILM and will be seen in preretinal space. DR.93 This is attributed to the fact that parafoveal
OCTA has emerged as an effective tool to differ- capillary non-perfusion in DCP may be an early
entiate between IRMAs and NVs.82 Different sign of DR. VD has been unaltered post

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 A Markan, A Agarwal et al.

Fractal dimension
Fractal analysis analyzes the microvascular altera-
tions and the geometric alterations of the retinal
vasculature. Fractal dimension in both the SCP
and DCP has shown a significant decrease in dia-
betic eyes compared with normal control sub-
jects. However, the metric does not vary among
different severity levels of DR.99

There are some limitations with use of OCTA.

The field of view with OCTA is smaller as
compared with available FFA platforms. Even
with the introduction of widefield OCTA of
12 mm × 12 mm scans, they are still not compara-
ble with ultrawide field FFA/indocyanine green
angiography (ICGA).83 Although this issue has
been overcome with montage OCTA using
12 mm × 12 mm scan, issues such as increased
acquisition time and misalignment of images are
Figure 5. Optical coherence tomography angiography
(OCTA; 3 mm × 3 mm scans) of two subjects with still a problem. Second, limitation with OCTA is
diabetic macular edema is shown. Patient #1 that it is unable to assess the dynamic characteris-
shows areas of capillary dropouts (yellow asterisks) tics of flow velocity. Third, problems like motion
in the superficial retinal plexus (a) along with and projection artifacts are commonly encoun-
increased intercapillary spacing and an irregular tered while analyzing the images.100
foveal avascular zone (FAZ; yellow dashed circle).
On the deep retinal capillary plexus (b), these
areas of capillary dropouts are better visualized.
Patient #2 shows increased intercapillary spacing 3D OCTA analysis
involving the central macula (yellow dashed square) Borrelli and colleagues101 have recently described
in the superficial capillary plexus (c) and multiple in vivo rotational 3D analysis of microaneurysms
microaneurysms in the en face scan of the deep using OCTA. Both OCT and OCTA provide
capillary plexus (d). structural cross-sectional and en face imaging of
the retina, respectively. This two-dimensional
(2D) visualization cannot provide information
dexamethasone injection in both SCP and DCP. about the origin, orientation, and location of
However, VD in choriocapillaris tended to microaneurysms within the retina. This short-
increase post treatment.94 coming can be overcome by using 3D analysis of
microaneurysms on OCTA. Overlapping anat-
omy and vessel foreshortening can be tackled by
Intercapillary spacing rotating these images on their three axes. Using
Studies have shown intercapillary spacing as more 3D visualization, it has been shown that microa-
sensitive parameter than VD and FAZ to detect neurysms are associated with two vessels and not
early capillary dropouts or areas of non-perfusion.95 at the vascular junctions. Besides, different mor-
phological varieties of microaneurysms can be
seen using 3D visualization.
FAZ
Quantitative measurements of FAZ on OCTA Zhang and colleagues102 have described an auto-
can be done using various indices like axis ratio, mated 3D shape modeling framework to obtain
FAZ area, acircularity index, perimeter, and high-quality 3D vessel representation using
area.96 Larger FAZs (both acircularity index and OCTA. The authors have used advanced surface
axis) have been seen on deep plexus slab in OCTA reconstruction models to visualize retinal micro-
scans of patients with diabetes compared with vasculature. It is possible to have a smooth repre-
normal irrespective of DR (Figure 5).97 Also sig- sentation of vessel tortuosity and allows clear
nificantly larger FAZ has been documented in delineation of large and small caliber vessels in a
eyes with DRIL as compared with eyes with no 3D view. Such analyses provide a very precise
DRIL.98 measurement capabilities. Furthermore, Borrelli

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Therapeutic Advances in Ophthalmology 12

Figure 6. Fundus autofluorescence (FAF) findings in a patient with diabetic maculopathy are depicted in the
figure. High signal of autofluorescence in panel (a) corresponds to diabetic macular edema (b), compared with
lesser autofluorescence (c) with no diabetic macular edema in the fellow eye (d).

and colleagues have quantified DME using 3D Also, intraretinal cysts in DME unmask the under-
vascular volume and perfusion densities in lying RPE by displacing the luteal pigment in the
patients with DR. The authors observed highest fovea centralis. This prevents the normal blockage
area under the receiver operating characteristic of foveal FAF signal at the level of retinal intrareti-
curve using 3D perfusion density, and concluded nal cysts (Figure 6).109,110 Significant FAF altera-
that such an analysis can be reliable and tions have been described in diabetic patients
efficacious.103 without retinopathy.109 FAF has been shown to
have sensitivity and specificity of 81% and 69%,
respectively, in detecting CME.110
Fundus autofluorescence biomarkers
Fundus autofluorescence (FAF) is a rapid, non- A number of studies using short wavelength FAF
invasive imaging technique that may give new have reported increased FAF signal in patients
insights into the evaluation of DME. Short- with DME.106,111,112 Various patterns of FAF have
wavelength FAF derives its signal mainly from been described, such as a single cyst of increased
lipofuscin in the RPE.104 Long wavelength auto- FAF, multi-cystic FAF, or combined single- and
fluorescence or near-infrared (NIR) FAF derives multi-cystic FAF. FAF was found to correlate bet-
its signal from melanin, which is present in RPE ter with OCT patterns and central field microper-
and choroid.105 Melanin accumulates in the api- imetry rather than with visual acuity.106 An
cal parts of the RPE cells and is thought to be increased FAF signal (hyper-autofluorescence) is
protective of the RPE. associated with worsening visual acuity and an
increase in the macular thickness on OCT.113 In
In DR, local ocular inflammation and oxidative patients with DME, the pretreatment baseline
stress lead to increased amount of lipofuscin and degree of foveal FAF signal might be helpful in
decreased amount of lutein and zeaxanthin in the predicting macular cube average thickness in
macula.106,107 This is responsible for increased patients undergoing treatment with intravitreal
FAF signal in subjects with DME. In addition, anti-VEGF in the short term.112 Thus, apart from
activation of microglia in diabetes could cause oxi- assessing the health of the RPE, FAF may have
dation of proteins and lipids.108 Histologic studies several applications in subjects with DR and
have found lipofuscin to accumulate in microglia. DME.

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 A Markan, A Agarwal et al.

Summary and conclusion 3. Jenkins AJ, Joglekar MV, Hardikar AA, et al.
Recent advancements in imaging techniques have Biomarkers in diabetic retinopathy. Rev Diabet
allowed a multimodal approach in diagnosis and Stud 2015; 12: 159–195.
management of various retinal diseases. With the 4. Zur D, Iglicki M, Busch C, et al. OCT
advent of SD and SS OCT, microstructural biomarkers as functional outcome predictors
details of retina and choroid can be easily appreci- in diabetic macular edema treated with
ated. CVI is a recently introduced imaging bio- dexamethasone implant. Ophthalmology 2018;
marker to study the choroidal vascularity on 125: 267–275.
OCT. Introduction of widefield FFA allows to 5. Classification of diabetic retinopathy from
examine peripheral vascular abnormalities in DR. fluorescein angiograms. ETDRS report number
Peripheral capillary non-perfusion areas can be 11. Early Treatment Diabetic Retinopathy Study
selectively targeted in cases of recalcitrant DME. Research Group. Ophthalmology 1991; 98(5
OCTA being a non-invasive, dyeless procedure Suppl.): 807–822.
clearly delineates the abnormal retinal vascula- 6. Sun JK, Lin MM, Lammer J, et al.
ture and non-perfusion areas. Introduction of Disorganization of the retinal inner layers as a
wider scans helps detecting vascular abnormali- predictor of visual acuity in eyes with center-
ties involving the peripheral retina. Finally, FAF involved diabetic macular edema. JAMA
has been studied recently in DME and may help Ophthalmol 2014; 132: 1309–1316.
to prognosticate and monitor the disease progres- 7. Radwan SH, Soliman AZ, Tokarev J, et al.
sion and response to treatment. These imaging Association of disorganization of retinal inner
techniques may help in the detection of subclini- layers with vision after resolution of center-
cal disease and retinal vascular changes, even involved diabetic macular edema. JAMA
before clinically detectable changes, or develop- Ophthalmol 2015; 133: 820–825.
ment of visual symptoms.
8. Nicholson L, Ramu J, Triantafyllopoulou I, et al.
Diagnostic accuracy of disorganization of the
Acknowledgements retinal inner layers in detecting macular capillary
We would like to acknowledge the efforts of Mr. non-perfusion in diabetic retinopathy. Clin Exp
Arun Kapil, Mr. Sushil Bhatt, and Mr. Nitin Ophthalmol 2015; 43: 735–741.
Gautam who helped in the acquisition of images
9. Balaratnasingam C, Inoue M, Ahn S, et al.
in our case.
Visual acuity is correlated with the area of the
foveal avascular zone in diabetic retinopathy and
Conflict of interest statement retinal vein occlusion. Ophthalmology 2016; 123:
The authors declared no potential conflicts of 2352–2367.
interest with respect to the research, authorship,
and/or publication of this article. 10. Das R, Spence G, Hogg RE, et al.
Disorganization of inner retina and outer retinal
morphology in diabetic macular edema. JAMA
Funding Ophthalmol 2018; 136: 202–208.
The authors received no financial support for the
research, authorship, and/or publication of this 11. Joltikov KA, Sesi CA, de Castro VM, et al.
article. Disorganization of retinal inner layers (DRIL)
and neuroretinal dysfunction in early diabetic
retinopathy. Invest Ophthalmol Vis Sci 2018; 59:
ORCID iD
5481–5486.
Vishali Gupta https://orcid.org/0000-0001-
8216-4620 12. Uji A, Murakami T, Nishijima K, et al.
Association between hyperreflective foci in the
outer retina, status of photoreceptor layer, and
visual acuity in diabetic macular edema. Am J
Ophthalmol 2012; 153: 710–717.
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