Optical Coherence Tomography and Clinicopathological Correlation for Understanding the Pathogenic, Clinical, and Prognostic Implications in Age-Related Macular Degeneration

Abstract

Optical coherence tomography (OCT) has emerged as a pivotal imaging modality in elucidating the pathogenic, clinical, and prognostic implications of age-related macular degeneration (AMD). This review examines the utility of OCT in providing high-resolution, cross-sectional imaging of retinal structures comparable to an in vivo histopathology. Recent histopathological correlations with OCT have enabled the precise characterization of AMD extracellular lesions, improving the interpretation of several OCT signatures. By correlating OCT findings with clinicopathological features, a deeper understanding of the underlying pathophysiology of AMD is achieved, facilitating early detection, risk stratification, and therapeutic decision making. Furthermore, OCT-derived biomarkers offer valuable insights into disease severity, response to treatment, and prognostic outcomes, thereby enhancing patient care and optimizing visual outcomes.

1. Introduction

Optical coherence tomography (OCT) is a non-invasive imaging modality in ophthalmology that enables cross-sectional views of the retina with high resolution comparable to histopathological specimens to be obtained [1]. The resolution of the commercially available OCT devices permits an accurate distinction of the vitreous anatomy and retinal layers and their structural alterations, as well as the choroid [2,3,4,5]. The identification of OCT features corresponding to pathological alterations remains a key research priority for several reasons. Firstly, OCT technology is widely utilized in clinical practice due to its high resolution, non-invasive nature, and the speed and ease of the procedure. Additionally, the integration of eye-tracking and image registration capabilities enables the precise monitoring of lesions over time, allowing for an accurate assessment of disease progression or response to treatment. Moreover, OCT has become a leading imaging technique in clinical trials for age-related macular degeneration (AMD), where several measure outcomes are essential to evaluate treatment response [6,7,8,9,10,11]. OCT was also used in the diagnosis of systemic disorders [12,13]. More recently, the evolution of this technology has allowed for the expansion of research in the field of artificial intelligence, using OCT biomarkers in predictive and prognostic models [14,15,16,17].

OCT has become a fundamental part of AMD evaluation as it allows for a three-dimensional high-resolution assessment of the elementary lesions, an accurate evaluation of ultrastructural changes preceding the development of atrophy, and a precise longitudinal evaluation of the atrophic lesions’ evolution beyond clinical ability [18,19]. Likewise, OCT is essential in diagnosing and monitoring neovascular AMD, where the exudative changes are easily and precociously detected with greater precision and resolution compared to color fundus photographs and fluorescein angiography [20].

Several OCT biomarkers have been identified for the evolution toward neovascular and atrophic stages. These biomarkers included qualitative and quantitative features, as intraretinal hyperreflective foci, drusenoid pigment epithelial detachment, hypo-reflective drusen cores, increased drusen volume (≥0.03 mm³), drusen and hyperreflective foci distribution, outer nuclear layer (ONL) and outer retinal hyperreflective band (ORB) thinning, and choroidal thickness [21,22,23,24,25,26,27,28,29]. Clinicopathological correlations conducted on eyes imaged in vivo during life and examined ex vivo post mortem have significantly advanced the understanding of the anatomical landmarks, ultrastructure, and composition of several features. These findings have relevant implications for elucidating the pathogenesis of AMD. A direct comparison between ex vivo and in vivo imaging is highly dependent by death to preservation time and expert handling [30,31,32].

The present narrative review focuses on OCT biomarkers in vivo that have been associated with clinicopathological correlates on ex vivo studies, leading to a deeper understanding of the AMD pathogenesis. Such AMD biomarkers has revealed significant clinical, prognostic, and therapeutic implications, providing insights on the early prediction of disease progression into late-stage complications. Despite these relevant implications, the significance and recognition of such pathological biomarkers is often overlooked and considered futile. Therefore, this narrative review aims to summarize histopathological correlates of OCT biomarkers, describing their structural composition, clinical appearance, and significance but also providing evidence of their clinical and prognostic value in AMD.

2. Clinicopathological Correlations with OCT Features in Non-Neovascular AMD

2.1. Crystalline Deposits

Cholesterol clefts are identified within disciform lesions secondary to AMD on histopathological specimens [33]. Cholesterol clefts seen on histopathology correlate with hyperreflective plaques on near-infrared (NIR) and sub-retinal pigment epithelium (RPE) hyperreflective lines on OCT B-scans [34,35]. A direct clinicopathological correlation was performed on an 86-year-old woman with GA, revealing hyperreflective plaques on NIR and hyperreflective linear deposits parallel to BrM on in vivo OCT. Post-mortem histology identified these features as corresponding to cholesterol clefts in the context of avascular fibrosis [34]. The cholesterol crystallization requires supersaturation under specific physical conditions, likely within an aqueous environment, as observed in neovascular lesions [35]. However, the evidence of crystallization in non-neovascular AMD has been attributed to the replacement of soft drusen content as seen in avascular fibrosis, representing a biomarker of end-stage drusen lifecycle [34].

The peculiar appearance of cholesterol crystals on OCT B-scan co-localizing with highly reflective plaques on NIR—so-called hyperreflective crystalline deposits (HCD)—has been observed in eyes with non-neovascular AMD associated with outer retina and RPE atrophy (cRORA) [34,36] (Figure 1). Similar lesions were described in the context of regressing drusen as single or multilaminar hyperreflective lines [37], further corroborating the idea that crystallization is linked to a modification of drusen oily content seen in the end-stage lifecycle [34]. These crystalline structures exhibit high reflectivity, leading to saturation artifacts that the authors hypothesized may vary in conformation depending on the detection scheme. In swept-source OCT (SS-OCT), they appear as linear artifacts, while in spectral-domain OCT, they manifest as planar artifacts. The generation of such artifacts is likely dependent on cholesterol concentration and RPE integrity [38].

The detection of HCD on OCT has significant prognostic implications. These structures exhibit a dynamic nature, emerging during drusen maturation and undergoing changes such as fragmentation, conglomeration, and resorption. These processes ultimately lead to the development of outer retina and RPE atrophy, as well as atrophy growth and expansion [36]. Therefore, the recognition of HCD in the setting of non-neovascular AMD represents a biomarker of progression into geographic atrophy and significant lesion growth.

2.2. Calcified Structures

Drusen are hallmarks of AMD exhibiting different phenotypic variants; the glistening appearance within depigmented roundish areas has been found to be associated with calcified material surrounded by atrophic RPE [33,40]. This peculiar drusen appearance has been called by different terminologies, including regressing drusen, calcified drusen, ghost drusen, and refractile drusen [37,41,42,43]. On clinical examination, calcified drusen may present a peculiar glistening and/or chalky-white appearance, better recognized on NIR where the hyperreflectivity is more evident [43,44,45]. On fundus autofluorescence (FAF), the lesions presented mid-hyperfluorescence with a hyperfluorescent ring or hyperfluorescent ring lesions. These lesions corresponded on the OCT B-scan to a hyperreflective cap with hyperreflective spherules and a hyporeflective interior [10,41,44,46].

The refractile nature of drusen, as observed on color imaging, NIR, and OCT B-scans, reflects its physical composition. Calcific spherules, stratified with mineralized layers of varying densities, can create multiple reflecting surfaces that produce light reflection [43]. A direct clinicopathological correlation was performed on an 86-year-old patient suffering from GA, with the last in vivo follow-up performed 4 months before death. Post mortem imaging and histopathology have demonstrated that heterogeneous internal reflectivity within drusen (HIRD) on OCT correlates with calcified nodules composed of crystalline calcium phosphate, as seen in histopathology. Spherules differ in appearance and composition, being characterized by a heterogeneous needle-like crystal structure with increased electron density and composed of whitlockite. The specific physical composition of spherules causes the refractility and reflectivity on OCT [46].

Clinical characteristics and multimodal imaging features of calcified nodules should be distinguished from other similar phenotypes, including sub-RPE tubules, outer retinal corrugations, and outer retinal tubulations. Sub-RPE tubules (Figure 2a,aI) have been recently described as ovoidal structures with a hyperreflective border and hyporeflective interior located in the sub-RPE-basal lamina space in association with drusenoid pigment epithelial detachment (PED). The origin of this signature remains debated. It has been hypothesized to represent clusters of activated RPE cells with basal laminar deposits (BLamD) migrating toward Bruch’s membrane, though it also shares some similarities with calcified structures [47]. Outer retinal tubulations (ORT) also enter in differential with calcified structures (Figure 2b,bI); the main characteristics are discussed below (See Section 3.3). Outer retinal corrugations (Figure 2c,cI) have been described as curvilinear hyperreflective multiple formations within atrophic areas, likely representing residual BLamD [48].

Calcified drusen (Figure 2d,dI) is a common finding in non-neovascular AMD, with an estimated prevalence of 42.7% [42]. This finding is commonly detected in association with geographic atrophy (GA) or complete outer retina and RPE atrophy (cRORA) [41,42,49]. The risk of progression to atrophy in eyes with calcified nodules is supported by evidence showing that RPE cell death and migration occur alongside nodule formation, indicating a marker of agonal RPE [46].

2.3. Outer Retinal Tubulations

Outer retinal tubulations represent a degenerative process of the outer retina in response to RPE disruption. The term ‘tubulation’ originates from the branching, tubular appearance of these structures as they wind along the outer nuclear layer. ORTs are readily identified on OCT B-scans, where they appear as round or ovoid formations with a hyperreflective border and a hyporeflective interior (Figure 2b,bI) [50].

The original histopathological description of ORTs preceded the imaging characterization, describing interconnecting tubes of surviving photoreceptors in correspondence with fibrovascular disciform scars [51]. A clinicopathological correlation performed on a 90-year-old female with bilateral GA confirmed that the ovoid hyperreflective structures in the outer nuclear layer observed on OCT B-scans correspond to tubulations seen in histopathology. The reflectivity of the external wall was attributed to a combination of the external limiting membrane and mitochondria, while the reflectivity of the inner wall was constituted by cone photoreceptors [52,53]. Further clinicopathological characterizations have demonstrated different stages in the evolution of ORT, where Müller cells may represent the initial cellular response, sealing off photoreceptors from the RPE–Bruch’s membrane complex. The formation of ORTs in the context of atrophic RPE is preceded by varying arrangements of the external limiting membrane (ELM), ranging from flat to curved and then from reflected to scrolled [54,55].

ORTs are identified in the late stages of AMD, including both macular neovascularization (MNV) and GA. In cases of MNV, it is particularly important to distinguish ORTs from intraretinal fluid to avoid unnecessary treatment. The distinctive characteristics of ORTs include their hyperreflective border with hyperreflective material, exclusive location within the outer nuclear layer, and tubular structure, which can be better appreciated on en-face [50]. Moreover, ORTs tended to persist over time with a poorer visual outcome in MNV despite anti-VEGF treatment [56,57]. Despite the relative stability over time, ORTs undergo evolutive changes due to progressive cone degeneration. This degeneration within ORTs is marked by the loss of outer segments, followed by the disruption of inner segments, ultimately leaving only the ELM in the end-stage ORT, possibly with the involvement of Müller cells [58,59].

The early detection of ORT precursors can be an important element in predicting disease evolution and is perhaps helpful in future therapeutic strategies and clinical trials [55].

2.4. Hyperreflective Foci

Hyperreflective foci (HRF) are thought to represent a phenotypic expression of pathological changes in the RPE observed in AMD (Figure 1) [39]. Another hypothesis for the origin of HRF suggests that they may be associated with activated microglial cells, a possibility that appears to be more relevant to exudative disorders [60,61,62]. Several clinicopathological correlations have allowed a precise characterization in both atrophic and neovascular AMD [30,31,63]. In eyes with intermediate AMD, the presence and the number of HRF at baseline were independently correlated with GA development after a 2-year follow-up period [64]. The predictive value of HRF in intermediate AMD eyes for the progression to cRORA or GA has been demonstrated in several studies [21,64,65,66]. Additionally, the quantity of HRF demonstrated a stronger correlation towards GA progression [66]. The topographical distribution of HRF at 0.5 mm eccentricity with respect to the foveal center appears characteristic of eyes developing GA [27].

A legacy of RPE alterations has been described in GA, reflecting the progressive degeneration secondary to AMD [32]. The RPE fate in GA passes through different morphological changes, which include apoptosis (shedding) and transdifferentiation to migratory phenotypes with subducted cells abundant in the atrophic areas and dissociated cells that migrate horizontally, perhaps contributing to centrifugal expansion [67]. The topographical distribution of HRF is more pronounced at the border of GA where the lesion is progressing. HRF is also more concentrated in the areas where new lesions are developing [68]. A higher concentration of HRF at the junctional zone has been found to be associated with a faster progression, with a significant change in the local progression rate (+86.3%) [69].

2.5. Thin Double Layer Sign Revealing Thick Basal Laminar Deposits

The classical definition of a double-layer sign (DLS) refers to the separation between RPE and its basal lamina with Bruch’s membrane, which is seen as parallel hyperreflective bands interposed by a hyporeflective space. This signature was originally described as being associated with polypoidal neovascularization [70]. Recent clinicopathological correlations have demonstrated that DLS signature can also originate from non-neovascular thickened basal laminar deposits (BLamD) [71,72]. BlamD represents a hallmark of AMD directly correlating with RPE and photoreceptor degeneration [73]. On an OCT B-scan, a thin DLS is characterized by a single continuous zone of hyporeflectivity between RPE-BL and BrM, while a thick DLS presented an evident RPE–BL–BrM separation with a multilayered reflectivity [24], as visible in Figure 3. The hyporeflective band within the RPE–BL–BrMis typically less than 5 μm in normal eyes, with a median thickness of 4.3 μm, making its recognition impossible with commercial OCT devices. In AMD eyes, this band can reach a median thickness of 7.8 μm, ranging approximately between 5 and 10 μm in the central 1 mm region [74]. Clinical studies have reported an even greater thickness in eyes with rapidly progressing atrophy, measuring approximately 23.2–24.1 μm on average, with a range of 10–50 μm at the thickest point [75]. It has been estimated that when BLamD reaches a thickness of 20 μm, the RPE, even if still present, becomes dysfunctional [71].

Thickened BLamD has been hypothesized to be associated with phenotypic variants of GA with worse prognosis, exhibiting a correspondence with a diffuse-trickling appearance on FAF [75]. Similar findings have been described in eyes presenting pseudodrusen with diffuse macular atrophy, where the splitting between RPE-basal lamina and Bruch’s membrane tends to progressively thicken over time. As BLamD thickens, the RPE becomes progressively dysmorphic until cRORA is developed, often with residual subretinal hyperreflective material that likely represents persistent BLamD [18,76]. The deposition of thickened BLamD between the basal RPE-BL and the choroidal circulation reduces the permeability of the Bruch’s membrane, impairing metabolic exchanges and potentially contributing to progressive RPE damage and the expansion of atrophic lesions [73].

A protective effect of the DLS in foveal-sparing atrophic lesions has been hypothesized, but the existing literature on this topic remains inconclusive [24,77,78,79,80]. The different origins of DLS may explain the discrepancies in the results. In this context, a thick DLS may indicate the presence of type 1 MNV, while a thin DLS is more likely associated with thick BLamD and the progression of cRORA [81].

2.6. Choroidal Caverns or Choroidal Lipid Globules

Lipid globules were described in the human choroid and subretinal space, representing a clinicopathological correlate of lipid droplets, a physical form of cholesterol [35,82,83,84]. The original histopathological description demonstrated sudanophilic and osmophilic extracellular and extravascular globules located in the posterior choroid [83]. Although choroidal globules have been observed in healthy postmortem eyes, their potential in vivo correlation was originally described frequently within GA areas [82,85,86]. These structures were identified as hyporeflective angular cavities in the choroid on OCT B-scan, occupying either Sattler or Haller layers, and were initially hypothesized to represent non-perfused ghost vessels [85]. However, it has been proposed that these hyporeflective angular formations within the choroid may actually represent lipid globules. The authors explained the frequent co-occurrence of caverns with atrophic areas due to the absence of RPE in these regions that might facilitate their visualization. Moreover, the lack of photoreceptors in the areas of atrophy may play a role in reducing the demand for lipolysis, favoring the maintenance of such structures over time [82,86].

Choroidal caverns have been described in a myriad of different retinal pathologies [87,88,89,90,91,92], making this finding not specific to AMD. However, recognizing choroidal globules is clinically relevant in distinguishing these findings from other hyporeflective choroidal lesions that may need further investigation. Furthermore, the hypothesis behind the genesis of such structures may help elucidate the intricate pathogenic pathways and potential therapeutic strategies in AMD [82]. Figure 4 shows a choroidal cavern on OCT B-scan [89].

3. Clinicopathological Correlations with OCT Features in Neovascular AMD

3.1. Crystalline Deposits

In neovascular AMD, the aqueous environment necessary for the supersaturation of cholesterol crystals is created by the active exudation from neovascular lesions, which are characterized by hemorrhage and fluid accumulation [35,93]. In a cohort of neovascular AMD eyes, the prevalence of this signature, as estimated through OCT, was 7% [35]. The onion sign can be identified on OCT B-scans as multiple highly reflective lines in the sub-RPE-basal lamina, corresponding to large hyperreflective plaques on NIR [35,38]. On clinical examination, the plaques can appear as well-defined areas of yellowish refractile plaques or yellow-gray subretinal deposition with refractile foci [93]. Multilayered hyperreflective lines within a retinal (PED) on OCT B-scans corresponded to cholesterol clefts seen on histopathological correlates, showing persistence after years (Figure 5) [94].

A higher concentration of cholesterol within multilamellar stratified cholesterol crystals is likely responsible for more intense planar and linear artifacts observed on both SD-OCT and SS-OCT [38]. The presence of an onion sign has been described in association with type 1 MNV or polypoidal/aneurysmal type 1 neovascular lesion in the sub-RPE-BL compartment, often surrounded by sub-RPE fluid. All the cases reported exhibited active exudation despite treatment with anti-vascular endothelial growth (VEGF) agents [93,95]. The presence of multilaminar hyperreflective deposits in the sub-RPE space resembling an onion sign was also described in association with type 3 MNV. This signature can be detected early and identified before exudative changes at the onset of a type 3 MNV [96].

3.2. Subretinal Lipid Globules

Subretinal lipid globules (SLG) on OCT B-scan presented with peculiar characteristics reflecting their physical nature. Lipid globules described by Friedman on histopathology correspond to hyporeflective roundish structures producing a characteristic posterior hyperreflective tail [97,98]. The posterior hyperreflective tail has been attributed to a lensing phenomenon caused by lipid droplets under the OCT light beam, as demonstrated through a phantom experiment. This artifact, with reference to the focal plane of OCT, depends on oil-droplet localization [97]. SLG was identified at the border of type 1 MNV under VEGF treatment, being dynamic in nature with reabsorption and reappearance over the same neovascular lesion [97]. During anti-VEGF treatment, SLG may disappear and reappear even in the absence of other signs of exudation. Therefore, it has been hypothesized that the detection of SLG can serve as an early biomarker of disease activity [97,99].

SLG can be detected over de novo type 1 MNV without other exudative signs and even in the setting of intermediate AMD (prevalence of 13.3%) as an early biomarker of non-exudative MNV (Figure 6) [97,98]. The identification of SLG in intermediate AMD eyes is associated with an 87.5% probability of detecting NE-MNV. Furthermore, SLG associated with NE-MNV has been associated with lesion growth without the increase in the risk of neovascular conversion [98].

3.3. Double Layer Sign

DLS represents an OCT signature that may facilitate the early detection of type 1 MNV before the development of exudation [70,72,81,100,101]. Different terminologies were used to describe this feature, which includes shallow, irregular RPE elevation (SIRE) and flat, irregular PED [70,81,102]. However, the term DLS remains the preferred terminology, as it most accurately describes the clinical appearance of this signature [81]. The OCT-based definition for DLS included two distinct hyperreflective bands, with the upper band corresponding to the RPE–basal lamina complex and the lower band representing Bruch’s membrane (BrM) [70]. The hyperreflective bands presented an interposed hyporeflective line that corresponded to either a type 1 MNV beneath BLamD or a thickened extracellular matrix material on histopathological correlation (Figure 3B) [72]. The quantitative criteria adopted on the OCT B-scan define a DLS as separating RPE-BL from BrM, with low internal reflectivity and a height <100 μm. The length of the DLS was variably set at either >250 μm or 1000 μm [78,103].

The identification of a DLS on a structural OCT B-scan is a surrogate marker of subclinical type 1 MNV with good predictive power [101,103]. This signature demonstrated a sensitivity of 73–100% and a specificity of 84–92.1% in predicting subclinical type 1 MNV [98,101,103]. The diagnostic performance of DLS as a biomarker of type 1 MNV is superior to SLG with a higher sensitivity [98]. This signature can also reveal non-exudative MNV that may remain in a quiescent state for years before developing exudative changes [104,105].

3.4. Hyperreflective Foci in Macular Neovascularization

Hyperreflective foci in the setting of neovascular AMD are a crucial biomarker with significant implications. While most intraretinal and subretinal HRF originate from RPE cells migrating in response to ischemia, other cellular components, likely of inflammatory origin, may confer a risk for the development of MNV (Figure 7) [106]. It has been suggested that microglial activation plays a role in exudative disorders, where activated cells tend to migrate from the inner to the outer retina. These cells may appear hyperreflective on OCT B-scans as they become engorged with lipids, potentially differing in size, clustering, and motility compared to RPE cells [35,60,61,62]. Migrating RPE cells appear to follow Müller glia with a trajectory tracking the Henle fiber layer toward vessels in the deep capillary plexus [107]. RPE cells corresponding to HRF on ex vivo OCT can migrate intraretinally, as solitary cells or a swarm of multiple RPE cells; with some multinucleated cells, RPE cells can surround intraretinal vessels or mix with outer segments’ photoreceptor debris, developing vitelliform material [108].

HRF detected on a structural OCT B-scan can represent the phenotypic expression of nascent type 3 neovascular lesions [109,110,111,112]. The evidence of HRF preceding a type 3 MNV of several months can be detected in approximately half of the cases [111]. A typical precursor of a type 3 MNV is represented by HRF at the level of the deep capillary plexus overlying drusen or a drusenoid PED [113]. A direct clinicopathological correlation was obtained on a 90-year-old female with bilateral type 3 MNV [114]. The evidence of HRF within the outer nuclear layer, outer plexiform layer, or inner nuclear layer on OCT B-scans with a detectable flow signal on OCT angiography in the absence of intraretinal fluid represents distinctive features of a nascent type 3 MNV. The lesions may progress with a down growth towards the RPE and the sub-RPE space, developing exudative changes [109]. The recognition of HRF was more often associated with type 3 MNV, pseudodrusen, drusenoid PED, thinner choroidal thickness, and ARMS2 (rs10490924) risk variant [115].

Drusen growth and confluence with overlying HRF represented a phenotype associated with neovascular conversion [22,68,116,117]. The topographical co-localization of drusen within the foveal center with overlying HRF and peaking at 0.5 mm eccentricity can also represent a risk factor for the development of MNV [27].

4. Conclusions

The advent of high-resolution multimodal imaging has significantly improved the detection and visualization of AMD pathological signatures, facilitating optimal histopathological comparisons. In this context, OCT represents the ideal imaging technique for this scope, offering cross-sectional visualization of the retinal structures and revealing cellular and extracellular details in vivo. The advantages of OCT also include accurate follow-up through an eye-tracking system that allows for the high-quality monitoring of long-term evolution, and its noninvasive nature permits seriated examination in an easily accessible manner, which has contributed to its widespread use in clinical practice.

In recent years, clinicopathological correlations have offered a deep understanding of AMD pathogenesis and progression. Crystalline deposits and lipid globules represent different physical forms of lipid deposition in AMD with significant clinical and prognostic implications. The recognition of cholesterol crystals in the setting of non-exudative AMD has been linked with conversion to de novo cRORA and/or atrophic lesion growth. Lipid globules correspond to lipid droplets on histopathology. These biomarkers are useful in detecting subclinical exudative changes and, thus, the presence of a neovascular conversion. HRF may have various implications in predicting atrophic expansion, as well as neovascular lesion activity and the development of a nascent type 3 lesion. An important distinction should be made between avascular and vascular DLS. A thin avascular DLS can be correlated with thick BLamD, which can be associated with a worse phenotype of geographical atrophy characterized by a faster progression. Moreover, a thick DLS can be an early biomarker of type 1 MNV before the development of exudation.

In conclusion, clinicopathological correlates are crucial for identifying precursors of advanced disease, which can have important implications as study endpoints in clinical trials to identify future therapies. Further studies are encouraged to pursue further clinicopathological correlations and understand their clinical and prognostic implications.