REVIEW

Pearls and Pitfalls of Optical Coherence TomographyAngiography Imaging: A Review

Enrico Borrelli . SriniVas R. Sadda . Akihito Uji . Giuseppe Querques

Received: January 14, 2019 / Published online: March 13, 2019� The Author(s) 2019

ABSTRACT

Optical coherence tomography angiography(OCTA) has significantly expanded our knowl-edge of the ocular vasculature. Furthermore,this imaging modality has been widely adoptedto investigate different ocular and systemicdiseases. In this review, a discussion of thefundamental principles of OCTA is followed bythe application of this imaging modality tostudy the retinal and choroidal vessels. A propercomprehension of this imaging modality isessential for the interpretation of OCTA imag-ing applications in retinal and choroidaldisorders.

Keywords: Choriocapillaris; Choroid; Imageanalysis; Optical coherence tomographyangiography; Retinal vessels

INTRODUCTION

Optical coherence tomography angiography(OCTA) is an emerging noninvasive imagingtechnique which is able to rapidly produceangiographic images of the eye [1, 2]. The basicconcept of OCTA is to detect the movement ofblood within vasculature as an intrinsic contrastagent. To obtain this, several repeated OCTB-scans are performed in the same location andare compared in order to detect differences, ordecorrelation, in the blood flow signal.

OCTA is mainly used to visualize the retinaland choroidal vessels. The retinal vasculature iscomposed of three distinct retinal capillarylayers: the superficial (SCP), middle (MCP), anddeep capillary (DCP) plexuses (Fig. 1) [3, 4]. Inaddition, a fourth vascular layer, the radialperipapillary capillary plexus (RPCP) or nervefiber plexus (NFP), is mainly located within thesuperficial nerve fiber layer (NFL) surroundingthe optic disc. In addition, OCTA is efficaciouslyable to image the innermost part of the choroidwhich is named choriocapillaris (CC).

While OCTA has several potential advan-tages, this imaging technique is not withoutlimitations. Therefore, some issues should beconsidered during the analysis and

Enhanced Digital Features To view enhanced digitalfeatures for this article, go to https://doi.org/10.6084/m9.figshare.7807940.

E. Borrelli � G. Querques (&)Ophthalmology Department, San RaffaeleUniversity Hospital, Milan, Italye-mail: giuseppe.querques@hotmail.it

S. R. SaddaDepartment of Ophthalmology, David GeffenSchool of Medicine at UCLA, Los Angeles, CA, USA

S. R. SaddaDoheny Eye Institute, Los Angeles, CA, USA

A. UjiDepartment of Ophthalmology and Visual Sciences,Kyoto University Graduate School of Medicine,Kyoto, Japan

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https://doi.org/10.1007/s40123-019-0178-6

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interpretation of OCTA images, including someartifacts which may affect the images [1].

In this review, we will describe the basicprinciples of OCTA technology and theapproaches to quantify OCTA variables. Thisreview is thus intended to better guide scientistsand clinicians on how to interpret OCTAfindings.

This article is based on previously conductedstudies and does not contain any studies withhuman participants or animals performed byany of the authors.

OPTICAL COHERENCETOMOGRAPHY ANGIOGRAPHY

Overview on Technical Aspects

Structural OCT can acquire volumetric images ofthe retina by executing successive single B-scansat different retinal locations. Differently, OCTAdevices perform several repeated B-scans at thesame retinal location and the obtained struc-tural information is compared to detect signalchanges secondary to flowing erythrocytes(motion contrast). Furthermore, each B-scan iscomposed of several A-scans that are acquired atsequential positions to generate the B-scan. TheA-scan rate is dependent on the OCT instrument

and typically ranges between 70,000 A-scans persecond (spectral domain devices) and 100,000A-scans per second (swept source instruments)among commercially available devices. Of note,faster acquisition speeds are available in variousresearch instruments.

A crucial role in OCTA imaging is repre-sented by the interscan time, which representsthe delay between two B-scans repeated at thesame retinal location. Therefore, while a shorterinterscan time may decrease sensitivity tomotion, this also reduces the occurrence ofparasitic eye motions which can confound themotion signal [1].

Three different methodologies may be usedto detect motion contrast using OCTA devices,as follows: (1) phase-based, (2) amplitude-based,and (3) complex amplitude-based, in which theOCTA algorithm uses both phase and amplitudeinformation. The OCTA signal is normalizedbetween 0 and 1. The interscan time and thebackground noise determine the slowestdetectable flow. Erythrocytes must move a suf-ficient distance within repeated B-scans to bedetected. In addition, differences in signal mustbe higher than the background noise to bedetected as flow. This causes an intrinsic limi-tation to current OCTA technology which maynot be able to distinguish the absence of flowfrom slow flow below a detectable thresholdrange [1].

OCTA may be captured with spectral domainOCT (SD OCT), which in commercial devicesemploys a wavelength at around 840 nm, orwith swept-source OCT (SS OCT), which uses alonger wavelength (* 1050 nm).

Visualization of the OCTA Images

While OCT is considered a cross-sectionalimaging modality, OCTA images are mainlydisplayed with en face visualization. Using thesegmentation of the volumetric OCTA scans atspecific depths, the flow data within any slabare summed or projected into a two-dimen-sional en face image that can be viewed andstudied.

The generation of two-dimensional OCTA enface images may be obtained using different

bFig. 1 Optical coherence tomographic angiographic imageof the macula of a healthy subject. The retinal vascular-ization at the macula includes four different plexuses: thesuperficial (SCP—second line from top), middle (MCP—third from top), and deep (DCP—second from bottom)retinal capillary plexuses, and choriocapillaris (CC—bottom). OCTA images are mainly displayed with en facevisualization (images on the right) which is obtained bysegmenting the volumetric OCTA scans at specific depths(indicated with white arrows). Using this strategy, the flowdata within any slab, whose boundaries are red in the leftimages, are summed or projected into a two-dimensionalen face image that can be viewed and studied. Theseboundaries follow predefined layers which can be differ-entiated on the basis of reflectivity, texture, or otherattributes (top row). The layers are: (i) the inner limitingmembrane (ILM), (ii) the inner border of the innerplexiform layer (IPL), (iii) the outer border of the outerplexiform layer (OPL), and (iv) Bruch’s membrane (BM)

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methodologies, including the ‘‘maximal inten-sity projection’’ approach which projects themaximal intensity pixel from each column ontoa flat two-dimensional summary. While theadvantage of this strategy is that it has moresensitivity for detecting smaller vessels andsubtle flow, it may also yield more noise.Another approach to obtain two-dimensionalOCTA images is through use of an ‘‘averageintensity projection,’’ which is less susceptibleto noise but may be less sensitive for smallvessels.

Of note, the en face visualization of theOCTA images is susceptible to segmentationartifacts, especially in eyes with retinal andchoroidal disorders [5].

Comparison with Previous Dye-BasedImaging Techniques

Fluorescein angiography (FA) and indocyaninegreen angiography (ICGA) have traditionallybeen considered the gold standard for clinicalassessment of the retinal and choroidal vascu-lature in vivo. Even though dye administrationis generally safe in patients, serious allergicreactions may occur, and these techniques areconsidered invasive and require considerablepatient cooperation. In contrast, OCTA pro-vides noninvasive assessment of the retinal andchoroidal vasculatures [6]. Moreover, OCTA hasthe additional advantage of depth resolutionwith improved visualization of the deeper vas-cular plexuses. In addition, both FA and ICGAhave significant limitations in the assessment ofthe choroid [2]. Despite these limitations, dye-based imaging provides other advantagesincluding dynamic estimation of dye transitand dye leakage in diseases affecting the blood-retinal barrier (e.g. diabetic retinopathy).

LIMITATIONS AND ARTIFACTS

Different Artifacts may Alterthe Visualization of OCTA Images

Low signal strength may significantly affect thequality of the OCTA images. The ratio between

the signal (signal-to-noise ratio), which is theinformation component dependent on theimaged tissue, and the non-signal component(or noise), is a mathematical relationship whichmay influence the image quality. As an exam-ple, media opacities may reduce the signal-to-noise ratio by decreasing the OCT signal. Adecreased signal-to-noise ratio leads to anadjustment of the grayscale range with a con-sequent increase in image noise and artifacti-tious OCTA signal. Averaging of multiple enface OCTA images may reduce noise andimprove the vessels’ continuity, as well as sig-nificantly improve qualitative and quantitativemeasurements [7, 8]. False OCTA signals may bealso generated by a patient’s movements [9].Eye tracking systems have significantlydecreased the impact of these artifacts in OCTAimaging.

Segmentation errors represent a significantand recurring OCTA artifact [5, 9]. As discussedabove, en face OCTA images are displayed bylocating two boundaries throughout the retinaland/or choroidal structure and vessels aretherefore visualized using different availableprocesses. These boundaries may be set byselecting predefined layers which can be differ-entiated on the basis of reflectivity, texture, orother attributes. The main problem with thisstrategy is that these parameters used to differ-entiate retinal and choroidal layers may be sig-nificantly modified in pathological conditions[5]. As an example, the automatic segmentationof vascular layers may be challenging in eyeswith myopia, even in the absence of chori-oretinal complications. Of note, many OCTAdevices employ software enabling manual cor-rection of segmentation errors and propagationof corrections throughout multiple B-scans, butthis process may be extremely time-consuming.

Projection artifacts (decorrelation tails;Fig. 2) may affect the visualization of the deepervascular layers (ICP, DCP, and CC). In order toimage the deeper structures, the OCT beampasses through the retinal layers, and vesselsincluded in more superficial layers may be fal-sely detected in the deeper layers, even in theabsence of real flow in these slabs. Since thetransmitted light diffuses through flowingblood, the deeper layers are thus reached by

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fluctuating light which can create a decorrela-tion between two successive B-scans at the samelocation. Several strategies have been evolved tolimit projection artifacts [10–12]. One of theeasiest strategies to limit projection artifacts isto subtract the SCP OCTA en face image fromthe deeper layers [10, 11, 13–15].

A shadowing artifact occurs when the OCTbeam is attenuated or blocked, thereby imped-ing its passage to the deeper layers of the reti-na/choroid. This may occur due to the presenceof different structures, including vitreous opac-ities, hemorrhage, and drusen. SS OCTA systemshave partially overcome this limitation, since alonger wavelength limits attenuation frommedia opacities and improves penetration intothe deeper retinal and choroidal layers [2].

OCTA is still limited in the visualization ofthe larger vessels of the choroid. This limitationis secondary to the signal attenuation due toscattering by the retinal pigment epithelium(RPE) and by the CC vessels [2]. As a result,choroidal vessels are typically displayed as sil-houettes with complete loss of signal at greaterdepths. These limitations have been partiallyresolved with ultrahigh-speed SS OCTA instru-ments [16, 17]. An improved visualization ofthe medium-sized choroidal vessels may also beobtained in highly myopic eyes using the pro-jection artifacts in the sclera [18].

OCTA Quantitative Metrics

While quantifiable metrics applied to imagesobtained using different OCTA devices weredemonstrated to be not comparable [19] andquantification is strictly dependent on theOCTA en face image’s size [20], different studieshave used quantitative variables to quantify theretinal and choroidal vasculature with OCTA.

OCTA en face images are composed of pixelswhich may be in a grayscale range of possiblevalues from 0 to 255. In order to obtain quan-titative analysis, a threshold may be applied tothese images. Thresholding is used to createbinary images [in which pixels over the appliedthreshold are displayed as white (or black) andpixels falling under the threshold are shown asblack (or white, respectively)] from grayscaleimages. Moreover, thresholding may beobtained using different methodologies thatcan significantly influence the final quantifica-tion [21]. The binarized OCTA images may bealso skeletonized, in order to obtain an image inwhich vessels are visualized as tracings of 1 pixelin width [22].

Binarized and skeletonized OCTA images(Fig. 3) may be used to obtain different quanti-tative metrics:• The perfusion density can be calculated as a

unitless proportion of the number of pixelsover the threshold divided by the total

Fig. 2 Example of projection artifacts. Superficial capillaryplexus (SCP—left image) and deep capillary plexus(DCP—middle image) from a healthy subject anddemonstrating the presence of projection artifacts from

major SCP vessels on the DCP image. Projection artifactsfrom superficial retinal vessels are subtracted and replacedwith dark versions of these vessels (right image)

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number of pixels in the analyzed area on thebinarized image.

• The vessel length density is defined as the totallength of the perfused vasculature divided bythe total number of pixels in the analyzedarea on the skeletonized image.

• The vessel diameter index, which representsthe average vessel caliber, may be calculatedby dividing the total vessel area in thebinarized image by the total vessel lengthin the skeletonized image [23].

Fig. 3 Main OCTA metrics to quantify the retinal andchoroidal vasculature. The OCTA images can be thresh-olded to obtain binarized and skeletonized images whichare utilized to provide a quantitative analysis. Thresholdingis used to create binary images from grayscale images, in

which pixels over the applied threshold are displayed asblack, and pixels falling under the threshold are shown aswhite. In this example, a MaxEntropy and Phansalkarthresholds were used to binarize retinal and CC vessels,respectively

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• The fractal dimension, which is a mathemat-ical parameter that describes the complexityof a biological structure [24].Previous studies on the CC have investigated

the distribution, size, and percentage of flowvoids (also called signal voids), which are smalldark regions that alternate with granular brightareas, the latter thought to represent CC flow,and the former more likely to be secondary toCC vascular dropout or flow reduction[2, 25, 26].

EVALUATION OF THE VASCULARLAYERS WITH OCTA

The Retinal Vessels

OCTA has significantly expanded our knowl-edge on the organization of the retinal vessels.The retinal vascularization may be separatedinto the following plexuses: the SCP, the MCP,the DCP, and the RPCP [27–30]. The RPCP isunique in that it is mainly located within thesuperficial nerve fiber layer surrounding theoptic disc.

Since the introduction of OCTA, a number ofmodels have been proposed regarding connec-tions among the retinal vascular plexuses[29–32].

The SCP is a dense meshwork of vesselsaccommodated in the retinal ganglion cell layer(GCL) [18]. The small capillaries arise from SCParterioles and drain into SCP venules. The GCLslab enhances visualization of the capillary-freezones around the arteries and arterioles andhelps differentiate arterial and venous systems[32]. Using OCTA, SCP arterioles were demon-strated to be located more superficially than SCPvenules [30]. The SCP capillaries were shown tobe arranged in a centripetal pattern in themacula and to converge on the parafoveal cap-illary ring [29]. In the periphery, the superiorand inferior circulations converge in an inter-laced comb pattern [29].

The SCP capillaries are connected with theother two layers of vessels at the inner plexi-form layer/inner nuclear layer (IPL/INL) border(MCP) and inner nuclear layer/outer plexiformlayer (INL/OPL) border (DCP). While the MCP

colocalizes with the bipolar cell processes andamacrine cells, the DCP colocalizes with hori-zontal cells. These two plexuses are thus close tothe high-oxygen-demand synapses of the IPLand OPL, respectively. The capillaries of theMCP and DCP have a lobular configurationwithout directional preference within theirlaminar planes [29]. Because the MCP and DCPconsist of capillaries of uniform size and areseparated by only a small distance, they areoften grouped together as the deep vascularcomplex (DVC).

Around the foveal avascular zone (FAZ),these three retinal capillary plexuses convergeto form a single parafoveal capillary loop andcollectively define the borders of the FAZ [29].Although some OCTA studies have separatelyinvestigated the FAZ area in different retinalcapillary plexuses, recent papers have opted toassess this measurement in a single segment(whole retinal thickness). This choice was dueto histologic and technical reasons: (1) there is astrong body of evidence suggesting the retinalplexuses merge at the edge of the FAZ, whichmay be thus considered a singular structurethroughout the entire foveal thickness [33]; and(2) assessing the FAZ size at different segmentsmay lead to increased variability of measure-ments [34]. The FAZ size was demonstrated toincrease with age [35, 36] and disease (e.g.myopia, hypertension) [37, 38].

Recently, using higher-axial-resolutionOCTA technology, Nesper and Fawzi [30] iden-tified distinct vascular connections from large-caliber arterioles and venules in the SCP to eachof the three retinal capillary plexuses in healthysubjects. This organization accounts for thehigh oxygen demand of the plexiform layersthat need highly oxygenated (arteriolar) bloodin the MCP and DCP. Furthermore, they pro-vided further evidence for the presence of vor-tices in the DCP, these draining into conduitswhich are straightly connected to venules in theSCP. Interestingly, collateral vessels crossing thehorizontal raphe were identified at the DCPlevel. These crossing vessels were speculated torepresent an alternative pathway of least resis-tance in the event of occlusive diseases. Nesperand Fawzi’s observations would appear to sup-port the theory that each of the three plexuses

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has its own arteriolar supply and venular drai-nage, which would theoretically allow eachneurovascular unit to have independent controlof its vascular supply under physiologicconditions.

This parallel organization of the retinal cir-culation suggested by Nesper and Fawzi hasbeen challenged by Freund and Sarraf [31] whohave suggested a predominantly series-basedarrangement of the retinal circulation, with thebulk of the venous drainage of the retina origi-nating at the level of the DCP. The presence ofcollateral vessels in the setting of retinal venousocclusive disease at the level of the DCP, but notat the SCP, would appear to support thiscontention.

Recently, using a full-spectrum probabilisticOCTA with a novel algorithm for three-dimen-sional projection artifact removal (PAR), Hiranoand colleagues [39] investigated the retinalmicrocirculation. Since full-spectrum OCTAalgorithms improve axial resolution, theauthors provided important insights into thethree-dimensional architecture of the retinalvasculature. Remarkably, unlike the MCP andDCP, the location of the SVP significantly variedamong retinal regions. In all parafoveal regionstested, the SVP was featured by a small peak atthe NFL-GCL junction and a larger, broaderpeak within the GCL. Conversely, the perifoveahad only a single SVP peak. Furthermore, thispeak was closer to the ILM in the perifovealnasal, superior, and inferior quadrants. Theauthors presumed that this displacement wassecondary to the thicker NFL associated withthe arcuate and papillo-macular bundles inthese regions.

The nerve fibers in the retina are unmyeli-nated and they thus require large amounts ofenergy to maintain ion concentration gradients[40]. The retinal nerve fiber layer contains theradial peripapillary capillary network to supplyoxygen and metabolites to the optic nerveaxons. The RPCP is composed of long, straightvessels with few apparent feed points andanastomoses [32]. These capillaries arise fromperipapillary retinal arterioles, extend radiallyfrom the optic disc in parallel with the nervefiber axons, farthest along the temporal arcades[41].

Using widefield OCTA, Jia and colleagues[42] examined the RPCP flow characteristics inten eyes of ten healthy patients and illustratedan association between perfusion in this vascu-lar network and nerve fiber layer thickness. Theperfusion of these regions may thus serve as asurrogate measure for the metabolic activity ofthe ganglion cell axons.

The Choriocapillaris

Given that previous imaging techniques havesignificant limitations for the evaluation of theCC, OCTA has evolved into a key technologyfor investigation of the CC [2, 25, 26].

En face OCTA images of the CC are gener-ated by segmenting a thin slab with a thicknessof 10–30-lm starting at Bruch’s membrane. Inthese OCTA CC en face images, small darkregions (called flow voids or signal voids)[43, 44] alternate with granular bright areas, thelatter thought to represent CC flow, and theformer more likely to be secondary to CC vas-cular dropout or flow reduction [44]. Signalvoids seem to represent the inter-capillaryspaces which have been characterized in histo-logical studies on the CC [45].

However, histological images of the CC areslightly similar to the OCTA versions. Withmore advanced OCTA technology, we expect tohave an improved and more truthful visualiza-tion of the CC. However, an enhanced visual-ization of the CC network may even beobtained with commercially available devicesusing multiple en face image averaging [46].The registration and averaging of sequential enface OCTA images was shown to transform thepoorly defined granular appearance of the CCdisplayed with OCTA into a morphologic pat-tern that more closely represented the mesh-work pattern observed on histology. Notably,not only did averaging improve CC visualiza-tion, but more precise quantitative measures ofthe CC were obtained.

In the CC OCTA images, the number andsize of the signal voids have been shown tofollow a mathematical relationship of a powerlaw: log (number of signal voids) equals a scal-ing factor times the log (size of signal voids)

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plus a constant [47]. The presence of a powerlaw relationship presumes that the CC is char-acterized by a propensity for capillary segmentsto be affected in the vicinity of an alreadynonfunctional segment.

As explained above, OCTA is still limited inthe visualization of the medium- and larger-sized choroidal vessels. This limitation is mainlysecondary to scattering by the pigment in theRPE and by the vessels in the CC, and conse-quent signal attenuation. Recently, Maruko andcolleagues [48] used an SS OCTA device toimage the choroid in healthy subjects, andsubsequently employed a novel algorithm toimprove the visualization of the choroidal ves-sels. They demonstrated that removal of theprojection artifacts of CC can make the chor-oidal blood flow visible. Importantly, the chor-oidal blood flow area was correlated withchoroidal thickness.

CONCLUSIONS

OCTA is an evolving technology which hassignificantly enhanced the visualization of theretinal and choroidal vessels. However, majorlimitations still affect its widespread applica-tion. The employment of OCTA to study oculardiseases, along with advancements of thistechnology, will certainly expand our knowl-edge of retinal and choroidal disorders.

ACKNOWLEDGEMENTS

Funding. No funding or sponsorship wasreceived for this study or publication of thisarticle.

Authorship. All named authors meet theInternational Committee of Medical JournalEditors (ICMJE) criteria for authorship for thisarticle, take responsibility for the integrity ofthe work as a whole, and have given theirapproval for this version to be published.

Disclosures. Enrico Borrelli: none. SriniVasSadda: Allergan (C, F), Carl Zeiss Meditec (F),

Genentech (C, F), Iconic (C), Novartis (C),Optos (C, F), Optovue (C, F), Regeneron (F),Thrombogenics (C). Akihito Uji: Bayer (F),Novartis Pharma K.K. (F), Senju (F), Nidek (F),Canon (F). Giuseppe Querques: Allergan (S, C),Alimera (S, C), Amgen (S), Bayer (S, C), KHB (S),Novartis (S, C), Roche (S), Sandoz (S), Zeiss (S,C); Bausch and Lomb (C), Heidelberg (C). Giu-seppe Querques is also a member of Ophthal-mology and Therapy’s Editorial Board.

Compliance with Ethics Guidelines. Thisarticle is based on previously conducted studiesand does not contain any studies with humanparticipants or animals performed by any of theauthors.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any non-commercial use, distribution, and reproductionin any medium, provided you give appropriatecredit to the original author(s) and the source,provide a link to the Creative Commons license,and indicate if changes were made.

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