Relationship of Basal Laminar Deposit and Membranous Debris to the Clinical Presentation of Early Age-Related Macular Degeneration

Abstract

purpose. To correlate basal laminar deposit (BLamD) and membranous debris, including basal linear deposit (BLinD), with the evolution of early age-related macular degeneration (AMD).

methods. A clinicopathologic collection of 132 eyes with a continuous layer of BLamD was reviewed. The thickness and type of BLamD and the sites of membranous debris deposition were correlated with the clinical progression of the disease.

results. Two types of BLamD, termed early and late, were identified based on light microscopic appearance by using the picro-Mallory stain. The progressive accumulation of late type BLamD correlated well with increasing BLamD thickness, advancing RPE degeneration, poorer vision, increasing age, and clinically evident pigment changes. Membranous debris initially accumulated diffusely as BLinD, most eyes with BLinD and early BLamD remaining funduscopically normal. However, membranous debris also formed focal collections as basal mounds internal to the RPE basement membrane and as soft drusen external to the basement membrane. Eyes in which membranous debris remained confined to basal mounds belonged to older patients with poorer vision, whereas patients with soft drusen were younger and had better vision.

conclusions. The presence of BLinD and early BLamD define threshold AMD, which manifests clinically as a normal fundus. Although late BLamD correlates most closely with clinical pigment abnormalities, it is the quantity and sites of membranous debris accumulation that appear to determine whether the disease develops pigment changes only or follows the alternative pathway of soft drusen formation with its attendant greater risk of choroidal neovascularization (CNV).

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly, industrialized populations, ^{1 2} and the development of intervention strategies will depend on an understanding of the early stages of the disease. It is important, therefore, to describe the evolution of the two deposits pathologically significant to AMD, basal laminar deposit (BLamD) and membranous debris, to understand better the threshold at which aging becomes AMD and to correlate their further accumulation with changes in fundus appearance and visual acuity, 

BLamD is found between the basement membrane of the RPE and its plasma membrane and consists of basement membrane proteins and long-spacing collagen. ^{3 4 5} BLamD was originally used as a histopathologic marker for AMD grading, ⁶ its thickness correlating well with the degree of RPE degeneration, photoreceptor fallout, and vision loss. Subsequent reports confirmed that BLamD was the most prevalent histopathologic finding in early AMD eyes. ^{7 8} A late, amorphous form of BLamD has also been described and is associated with more severe RPE degeneration. ⁹  

The routine availability of transmission electron microscopy (EM) allowed the identification of basal linear deposits (BLinD), a deposit situated between the RPE basement membrane and the inner collagenous zone of Bruch’s membrane. ¹⁰ BLinD is composed primarily of membranous material and is specific for early AMD. ¹¹ In addition to forming this diffuse layer, the membranes may also be found in focal aggregations (i.e., as basal mounds between the RPE basement membrane and its plasma membrane) ⁹ and as soft drusen external to the basement membrane resulting from the buildup of BLinD. ^{7 10}  

The basal deposits are not directly visible on funduscopy, and hence their relationship to the clinical evolution of AMD is not well documented. The present study reviews a clinicopathologic series of 132 eyes in which diffuse basal deposits had been identified previously. ⁶ Although membranous debris in the BLinD can be demonstrated only by EM, the focal basal mounds and soft drusen can be identified by light microscopy, so that the respective influence each exerts on the progress of degeneration can also be assessed in histologic specimens. The type and thickness of BLamD are correlated with the sites of membranous debris accumulation and with the clinical findings. The threshold at which aging becomes early AMD is discussed. 

Methods

Patients and Eyes

In earlier studies a large clinicopathologic collection of aged eyes had been divided into histopathologic groups according to the appearance of the BLamD under the macula. ⁶ Briefly, eyes with no BLamD (group I) and patchy BLamD (group II) were considered normal. The eyes chosen for the present study were those with a thin (group III) or thick (group IV) continuous layer of BLamD, considered to represent the pathologic changes preceding advanced AMD (groups V and VI). A total of 132 eyes belonging to 75 patients (69 men and 6 women) aged 67 to 97 years at death (mean, 82 ± 8 years) met the inclusion criteria (Table 1) . 

All the patients had been examined clinically by one of us (SS), with follow-up ranging from 0 to 120 months (mean, 24 ± 32 months). Examination included best corrected visual acuity, direct funduscopy, and, unless unobtainable, fundus photography with a 30° fundus camera (Carl Zeiss Meditec, Inc., Dublin, CA). Fluorescein angiography was performed when appropriate. Eyes in which the fundus could not be adequately visualized or those that demonstrated other disease were excluded. The last examination ranged from 2 weeks to 75 months before death (mean, 19 ±17 months). 

The study was conducted in keeping with the tenets of the Declaration of Helsinki and approved by the University of New South Wales Human Research Ethics Committee. Written consent was obtained from all patients. 

Histopathologic Methods and Definitions

Eyes were either paraffin embedded for light microscopy (n = 107) or fixed for electron microscopy (n = 25) according to previously reported procedures. ^{6 10} For histopathology, serial sections 8 μm thick were cut horizontally through the disc and macula, and every 10th section was stained and examined. Three sections closest to the center of the fovea and 80 μm apart were examined with a microscope (Dialux; Leitz, Wetzlar, Germany). Objectives used were the APO 25× (NA = 0.65) and PL APO 40× (NA = 0.75). The microscope was calibrated by using a standard 0.01-mm objective micrometer graticule (Olympus, Tokyo, Japan), and measurements were obtained with an eyepiece graticule, one division equaling 2.8 μm with the 40× objective and 4.5 μm with the 25× objective. 

Histologically, BLamD was distinguished most readily with the picro-Mallory staining method, with which the early type showed faint anteroposterior striations and stained blue and the later type was hyalinized and stained red. BLamD was then graded according to two parameters: thickness and staining characteristics. Maximum BLamD thickness was recorded as thin if it was up to half the height of the normal RPE (≤7 μm) and thick if greater (>7 μm). ¹² BLamD staining was graded in the following degrees of progression: (1) blue-staining early type only (Fig. 1A) ; (2) patchy late BLamD appearing either as small, rounded inclusions approximately 4 μm in diameter lying within the early BLamD (Fig. 1C)or as larger nodular elevations occurring singly or in rows on the internal surface of the early BLamD, each nodule being overlain by a single RPE cell (Fig. 1D) ; (3) continuous late BLamD comprising segments ≥250 μm in length (Figs. 2B 2C) . On EM the earliest form of BLamD was fibrillar and was continuous with the original basement membrane of the RPE, but the predominant constituent of early BLamD was the banded form. This consists primarily of long-spacing collagen (Fig. 1B)accounting for the striations seen on light microscopy. Late BLamD had a more condensed structure and appeared to be produced in waves as the overlying RPE retracted (Fig. 2C , inset). 

Membranous debris was defined on EM as coiled membranes with a trilaminar appearance (Fig. 3)and was assessed by EM in 25 eyes (18 patients, mean age, 79 ± 7.14 years). The membranes often had a vesicular outline and may have contained lipids lost during processing, ¹³ so that the term “membranous” is used herein as an ultrastructural description only. BLinD was defined as a layer of this membranous debris lying between the RPE basement membrane and the inner collagenous zone of Bruch’s membrane. ^{7 10 11} The maximum number of layers of BLinD were recorded for each eye examined by EM. Basal mounds (Fig. 4)were defined on EM as pockets of membranous debris ¹¹ lying internal to the RPE basement membrane and early BLamD. In histologic sections, they appeared a washed-out, pale blue with picro-Mallory staining, were at least half the height of an RPE cell, and were mostly one to two RPE cells wide. For each eye, the maximum number of mounds in any one of the three sections through the macula was recorded. 

Soft drusen were defined as focal accumulations of the BLinD with sloping margins measuring over half the height of the normal RPE, up to 350 μm wide when confluent (Fig. 5) , and up to 500 μm wide in two cases with drusenoid detachment. Like basal mounds they stain blue with the picro-Mallory method, but unlike basal mounds, soft drusen lay below the RPE basement membrane. Drusen contents were recorded as predominantly membranous or predominantly granular, those with granular contents being regarded as regressing. 

Hard drusen (or nodular drusen ⁷ ) had a globular, hyalinized appearance and stained red with the picro-Mallory method. A few hard drusen are not considered part of AMD. One to two per section were seen in 85 study eyes. 

Histologic abnormalities of the RPE at the macula were graded as mild, moderate, or severe depending on the degree of irregularity, hypertrophy, and hyperpigmentation, or attenuation. 

Clinical Parameters

The best corrected visual acuity was converted to logMar (logarithm of the minimum angle of resolution) for statistical purposes. The funduscopic appearance of the macula was graded as: (1) normal, which included a few (<5) small (<63 μm) drusen; (2) multiple small drusen (<63 μm) involving an area >125 μm; (3) intermediate drusen (63–124 μm) with or without pigment; (4) large drusen (≥125 μm) with or without pigment; or (5) pigment abnormalities alone. 

Statistical Methods

Statistical analyses were performed on computer (SPSS for Windows, ver. 12.0.1; SPSS, Chicago, IL). Contingency tables of histologic versus clinical parameters were evaluated by the χ² test. Comparison of mean age and visual acuities was evaluated by Student’s t-test. Correlations between BLamD type, BLamD thickness, and histopathologic RPE abnormalities were performed by using Spearman’s correlation for nonparametric data. P ≤ 0.05 was considered statistically significant. 

Results

The clinical and histopathologic features of the study eyes are summarized in Table 1 . 

BLamD and RPE Changes

The RPE was not normal, even in eyes in which BLamD was exclusively of the early type, exhibiting a loss of uniformity and increase in pigmentation (Fig. 1A) . However, it was the amount of late-type BLamD that correlated positively with increasingly severe histologic RPE abnormalities (r = 0.435, P < 0.001), first appearing as patchy late BLamD in the form of inclusions (Fig. 1C)and then nodules (or nodular excrescences ¹⁴ ; Fig. 1D ). In the presence of severe RPE abnormality, late BLamD formed an unbroken layer exceeding 250 μm in length (Figs. 2B 2C)– termed continuous late BLamD herein and also described as diffuse thickening of the internal aspect of Bruch’s membrane. ¹⁵ The late form always lay on or near the internal aspect of the early type, closest to the base of the retracting RPE. 

BLamD in group III eyes formed a thin layer, ranging in thickness from 1.0 to 7.0 μm (3.2 ± 1.6 μm) and in 66 of 95 eyes (69%), was exclusively of the early type. Patchy, late BLamD was present in the remaining 29 group III eyes (31%). By contrast, continuous late BLamD was found in all 37 group IV eyes (100%) and ranged in thickness from 8 to 25 μm (10.7 ± 4.2). Overall, there was a strong positive correlation between BLamD thickness and the proportion of late type BLamD (r = 0.650, P < 0.001; Table 2 ). 

BLamD: Clinical Correlations

Clinically, 41 (62%) of 66 eyes with early BLamD had a normal fundus at the last clinical examination. Eyes with early BLamD alone were 10.4 times (95% CI, 4.4–24.6, P < 0.001) more likely to have a normal fundus than were eyes showing any late BLamD, clinical pigment changes being present in all those in which late BLamD was continuous. Only 10 eyes had clinical pigment changes without evidence of late BLamD, and 4 of these eyes were subsequently reclassified as adult vitelliform lesions. Increasing formation of late BLamD was associated with poorer vision, the mean logMar visual acuity for eyes with continuous late BLamD being 0.335 units worse than the other eyes (95% CI, 0.194–0.477, P < 0.001). Continuous late BLamD also occurred in eyes that were on average 3 years older (95% CI, 0.3–6 years, P = 0.013) than eyes with early BLamD (Table 2) . 

Membranous Debris: Localization and Correlation with BLamD

Coiled membrane fragments were found at several levels reflecting their presumed transit: blebbing from the basolateral RPE surface, scattered within early BLamD, traversing the RPE basement membrane to form BLinD, and in the inner and outer collagenous layers of Bruch’s membrane (Fig. 1B) . BLinD was present in all eyes examined by EM and ranged from 2 to 11 layers in thickness. 

A greater quantity of debris formed basal mounds and soft drusen. Because basal mounds were found only in the presence of BLinD and because they can be recognized in histologic sections, the mounds were considered a surrogate marker for the presence of BLinD at the light microscopic level. Membranous debris accumulation was limited to basal mounds in 44 eyes, whereas in 50 eyes, it extended to soft drusen formation. Although basal mounds were seen in the absence of soft drusen, soft drusen never occurred in the absence of basal mounds. 

Basal mounds increased in number as early-type BLamD thickened, but then plateaued with the formation of late BLamD. On EM there was also loss of some of their membranous contents. In addition, soft drusen in eyes with continuous late BLamD were predominantly of the granular type (12/15, 80%). This gradual reduction in membranous debris mirrored the progressive degeneration of the RPE and fallout of photoreceptors. 

Membranous Debris: Clinical Correlations

Although soft drusen as small as 20 μm were found histopathologically, they never occurred in the absence of larger drusen visible clinically. Thus, histopathologically detected soft drusen correlated very well with clinically observed intermediate and large drusen (r = 0.953, P < 0.001). In four eyes of two patients with normal fundi, intermediate-sized drusen were found on histologic examination. However, the interval between last examination and death was 5 and 3 years in these cases. There were also five eyes from three patients with multiple small drusen clinically, that were found to be soft drusen on histopathology. 

In group III, the fundus appeared normal in 28 (74%) of 38 eyes in which membranous debris was limited to BLinD, confirming that BLinD in conjunction with early BLamD represents threshold AMD. Basal mounds did not appear until early BLamD had thickened sufficiently, indicating that membranous debris and early BLamD continue to develop together. However, in 18 (75%) of 24 of group III eyes with basal mounds alone, the fundus remained normal, and it was not until patchy late BLamD appeared that most of the fundi exhibited abnormality. 

In group IV, all eyes had continuous late BLamD, and there was little further increase in membranous debris. In eyes in which the debris remained confined to basal mounds the mean segment length of late-continuous BLamD was 413 μm longer (95% CI, 87–740 μm, P = 0.015) than eyes with soft drusen. These eyes belonged to patients 6 years older (95% CI, 2–9 years, P < 0.001), and visual acuity was 0.402 logMar units worse (95% CI, 0.167–0.637, P < 0.001). Table 3summarizes the clinical findings of eyes in groups III and IV according to the amount of membranous debris present. 

Discussion

Zarbin ¹⁶ proposed that the biological changes associated with tissue aging, while present in AMD eyes, do not inevitably lead to AMD. The present study was undertaken to trace the evolution of BLamD and membranous debris, which have no counterpart in earlier life, to determine their relationship to aging and AMD. 

We found that the first appearance of BLinD coincides with a continuous layer of early BLamD. Because membranous debris is not found in eyes without BLamD (group I) and occurs only as occasional, isolated fragments in eyes with patchy BLamD (group II), ¹⁷ the presence of both BLinD and continuous early BLamD represents threshold early AMD and can be considered a continuum of normal aging. Also described as having “incipient AMD,” ^{11 18} most of these eyes were confirmed by us to have a normal fundus and good vision. 

The appearance of late BLamD signals severe RPE abnormality and corresponds to clinical pigment changes. Once produced, BLamD is remarkably resilient, persisting even in areas of geographic atrophy and in disciform scars. ⁹ BLamD is not lost during processing, remaining detectable using routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining and, although not specific for AMD, it is found in all eyes with the disease. These properties make it a useful and reliable histopathologic marker for AMD. In terms of the disease process, however, BLamD appears inert. Rather, it is the degree of membranous debris accumulation that appears to influence the course of disease. In the present study, the number of basal mounds increased as BLamD progressively thickened but, once RPE cells lost their ability to support the overlying photoreceptors, membrane production ceased. This was heralded by the appearance of continuous late BLamD and a loss of the membranous contents of the mounds. Soft drusen likewise became less membranous and more granular, interpreted as the onset of drusen regression. 

However, in some eyes membranous debris production did not progress beyond the formation of basal mounds. Even in the ninth decade, these eyes showed no histopathologic evidence of soft drusen, instead developing pigment changes associated with poor vision. Continuous late BLamD was always present in these eyes and in longer segments than in eyes with soft drusen, suggesting longstanding RPE dysfunction. By contrast, soft drusen sometimes occurred in relatively young eyes with good vision, before the appearance of continuous late BLamD. Clinically, intermediate and large drusen are known to increase in size, number, and confluence comparatively rapidly, ¹⁹ implying a rapid outpouring of membranes. However, the resultant thickening of BLinD is not uniform, causing it to develop undulations, the larger of which become visible as soft drusen (Fig. 4) . In these eyes, soft drusen do not appear to develop from an earlier stage when only basal mounds are present, i.e., soft drusen and basal mounds appear concurrently. 

Epidemiologic data support the concept of two pathways leading to the development of advanced AMD, depending on the amount of accumulated membranous debris. Eyes with a large amount of membranous debris, in the form of large drusen, are at highest risk of developing advanced AMD over a 5- or 10-year period, ^{20 21} particularly the blinding effects of CNV. ^{22 23 24 25} Eyes with pigment changes alone are at lower risk of developing advanced AMD over the same period, ^{20 21} consistent with an alternate and slower course of disease. 

This clinicopathologic study represents an overview of the basal deposits in the evolution of AMD, but makes no attempt to correlate with the clinical fundus grades or severity scales used in recent epidemiologic studies. It has two obvious limitations: first, the sometimes long interval between the last examination and death, with drusen a particular concern since the clinical appearance can change relatively rapidly. With a few exceptions, however, we found good correlation between histopathological soft drusen and clinical intermediate and large drusen, and the main study findings were unchanged by the exclusion of eyes with an interval longer than 36 months; second, differences in the postfixation method for EM prevented comparison of membranous debris with other recent studies of this tissue. ¹³ It should thus be emphasized that the terms BLinD, basal mounds, and soft drusen are pathologic descriptors of membranous debris locations, and that the biochemical nature of this material has not been completely defined. 

BLamD and membranous debris may be products of two distinct cell survival strategies of RPE under stress. Early BLamD can be thought of as excess basement membrane secreted by the RPE, a common strategy used by cells attempting to recover from injury, allowing them to remain attached to a tissue’s “scaffolding.” As BLamD thickens, it progressively separates the basal RPE surface from its original basement membrane and choroidal blood supply, exacerbating the metabolic insufficiency caused by decreasing the permeability of Bruch’s membrane. ^{26 27 28 29} The production of late-type BLamD signals a critical point in RPE damage at which the secreted basement membrane material becomes more condensed and, once this forms continuous segments, the RPE cells become hyperpigmented, enlarge, lose their microvilli, and round off. These phenotypic changes are initially accompanied by expression of the cytoskeletal protein vimentin. ³⁰ The RPE at this stage is severely compromised and can no longer support the photoreceptors. Eventually, affected RPE cells lose their anchoring to both the basement membrane and to adjacent cells and are shed into the subretinal space. ³¹  

The production of membranous debris, in contrast, may reflect another survival mechanism adopted by RPE under stress. Ultrastructurally, the earliest membranes appear to bleb from the basolateral surface of the RPE. Nonapoptotic membrane blebbing has been observed in cultured RPE cells subjected to oxidative stress ³² and is considered a nonspecific cellular response to ischemic, oxidative, or other injury which allows the expulsion of damaged cellular constituents. ^{33 34} Other studies show that membranous debris contains solid lipid particles, ¹³ and that dysregulated lipid trafficking by abnormal RPE may also contribute to its formation. ^{35 36} Because the appearance of BLinD coincides with a continuous layer of early BLamD, a threshold level of RPE injury (perhaps ischemic) may trigger the production of membranous debris. 

In summary, late BLamD occurs in eyes with clinical pigment changes and indicates a severely compromised RPE. It is membranous debris, however, that appears to influence the course of the disease (Table 4) . Eyes in which membranous debris accumulation is limited to basal mounds present with pigment abnormalities alone. These eyes eventually develop “primary” or “drusen-unrelated” ^{9 31} geographic atrophy. If there is a more rapid accumulation of debris, early AMD presents with intermediate or large drusen, is at greater risk of CNV ^{22 23 24 25} and earlier vision loss, and eventually progresses to “drusen-related” geographic atrophy. ³¹ These two pathways share a common threshold stage when both BLinD and a continuous layer of early BLamD are present (Fig. 6) . However, since most of these eyes are normal in fundus appearance and visual acuity, identifying at-risk eyes must rely on more sophisticated clinical tests, possibly combined with genetic screening. 

 Supported by National Health and Medical Research Council PhD Scholarship 008103 (SC).

 Submitted for publication April 20, 2006; revised September 22 and October 26, 2006; accepted January 17, 2007.

 Disclosure: S. Sarks, None; S. Cherepanoff, None; M. Killingsworth, None; J. Sarks, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Patient Characteristics

	Histopathological Group
		III (Thin Continous BLamD)	IV (Thick Continuous BLamD)
Number of eyes/patients	95/53	37/22
Mean ± SD	81 ± 8	84 ± 6
Age at death (y)
Range	67–97	74–92
Sex
M/F	49/4	20/2
Interval between last visit and death (mo)
Mean ± SD	21 ± 18	12 ± 8
Range	0–36	1–28
Total clinical follow-up
Mean ± SD	23 ± 32	28 ± 31
Range	0–120	0–99
Visual acuity (logMar)
Mean ± SD	0.276 ± 0.228	0.611 ± 0.401
Range	0.000–1.000	0.000–1.300
Fundus appearance
Normal	50	0
Pigment only	16	20
Drusen* only	18	0
Drusen* + pigment	11	17
Number of eyes examined by EM	21	4
BLamD thickness (μm)	3.2 ± 1.6	10.7 ± 4.2
BLamD type
Early only	66	0
Patchy late	29	0
Continuous late	0	37

*  Clinical drusen defined as intermediate or large drusen.

Figure 1.

 

Basal laminar deposit. (A) Early type BLamD forming a blue-staining continuous layer beneath the RPE ( ), up to half the height of an RPE cell. The fundus at age 67 had shown a few small soft drusen before death at age 71. (B) Electron micrograph illustrating changes between RPE and the choriocapillaris (CC). Early BLamD (bracket) lies internal to the RPE basement membrane (vertical arrows) and comprises mostly banded material resembling long-spacing collagen. Other phenotypes comprise a darker and denser material with an enveloping rim of pale material. Between the clumps of BLamD lie membrane fragments (horizontal arrow) that also form a layer external to the basement membrane, the BLinD ( ), and can be traced even into Bruch’s membrane. (C) Early BLamD containing small hyalinized clumps of the late type (arrows), stained red with picro-Mallory and found close to the RPE. The fundus at age 75 had shown early focal hyperpigmentation related to small soft drusen before death at age 76. (D) Further buildup of late BLamD formed nodular excrescences on the internal surface of the early type, with each nodule overlaid by a single RPE cell. The fundus had shown pigment changes and small soft distinct drusen 4 years before death at age 85. (A, C, D) picro-Mallory stain. Scale bar: (A) 25 μm; (B) 1 μm; (C) 20 μm; (D) 50 μm.

Figure 2.

 

Continuous late basal laminar deposit. (A) Eye of 79-year-old man photographed 2 years before death, showing ring of pigment clumps around foveal perimeter (arrow). (B) Section through the fovea of the eye illustrated in (A), showing a thick layer of late type BLamD. Pigment clumps in the fundus correspond to large, hyperpigmented RPE cells (arrow). (C) Higher magnification of (B). A thick, confluent layer of late BLamD lay on the internal surface of early type BLamD. Inset: Electron micrograph of late type, showing amorphous structure apparently laid down in waves, as grossly abnormal RPE retracts from Bruch’s membrane. Magnification, ×1200. (B, C) Picro-Mallory stain. Scale bars: (B) 200 μm; (C) 30 μm.

Figure 3.

 

Electron micrograph of membranous debris in the eye of an 81-year-old woman with normal fundus and 20/30 vision. Higher magnification (inset) shows two dark laminae enclosing a single electron lucent lamina (arrow), giving rise to a “trilaminar” appearance. Magnification, ×31,000; inset ×100,000.

Figure 4.

 

Basal mounds. (A–C) Section through the macula of a clinically normal eye of a 79-year-old man. Thin BLamD (arrowheads) contained several basal mounds that are histologically detectable as unstained spaces (arrows). Picro-Mallory stain. (D) Semithin section from an eye of an 80-year-old man with basal mounds ( ). BLinD appears as a narrow interval beneath the RPE and BLamD (arrow). Methylene blue and basic fuchsin. (E) Electron micrograph of a basal mound internal to the RPE basement membrane (arrows), in a patient with a fundus that had shown small drusen at age 85. ( ) BLamD. Scale bars: (A) 60 μm; (B, C) 50 μm; (D) 30 μm; (E) 5 μm.

Figure 5.

 

Membranous drusen. (A) Semithin section of the retina of a 75-year-old man, with clinical large drusen. Basal mounds of membranous debris (m) appear above BLamD ( ). BlinD has built up into soft drusen (d). Methylene blue and basic fuchsin. (B) Electron micrograph of a subclinical soft druse in the eye illustrated in (A), demonstrating membranous contents. ( ) Late BLamD overlaying early BLamD. (C) Fundus of fellow eye of the same patient at age 71, showing soft drusen measuring up to two vein widths. Scale bar: (A) 100 μm; (B) 5 μm; (C) 250 μm.

Table 2.

 

Subjects with BLamD

	BLamD
		Early Only	Patchy Late	Continuous Late
Clinical
Fundus appearance
Normal	62.1%	31.0%	0.0%
Pigment changes only	15.2%	20.7%	54.1%
Drusen* with pigment changes	16.7%	24.1%	0.0%
Drusen* only	6.1%	24.1%	45.9%
Mean Age (y)	80 ± 8	82 ± 8	84 ± 6
Mean visual acuity (logMar)	0.275 ± 0.242	0.280 ± 0.197	0.611 ± 0.401
Pathological
RPE abnormalities	Mild	Moderate	Severe
Membranous debris
BLinD, † (no. layers)	7.0 ± 2.3	6.0 ± 0.0	5.8 ± 2.1
Basal mounds	1.7 ± 2.5	2.7 ± 1.1	3.1 ± 1.9
Soft drusen	Membranous contents	Contents becoming increasingly granular
Eyes (n)	66	29	37

*  Clinical drusen defined as intermediate or large drusen

†  Demonstrated on EM.

Table 3.

 

Characteristics of Groups III and IV

	Membranous Debris	Number of Eyes	BLamD Thickness (μm)	BLamD	Fundus Appearance	n	Mean Age (y)	Mean VA (logMar)	Approx. Snellen Equivalent
Group III: thin continuous BlamD	BLinD only	38	2.5 ± 1.2	Early only 38	Normal	28	80 ± 8	0.245 ± 0.206	6/10; 20/30
					Patchy late 0	Pigment only	100
					Continuous late 0	Drusen
					Drusen + pigment	0
	Basal mounds only	24	2.9 ± 1.4	Early only 12	Normal	18	83 ± 6	0.273 ± 0.114	6/12; 20/40
				Patchy late 12	Pigment only	6
				Continuous late 0	Drusen	0
					Drusen + pigment	0
	Basal mounds + soft drusen	33	4.5 ± 1.6	Early only 16	Normal*	4	81 ± 8	0.311 ± 0.298	6/12; 20/40
					Patchy late 17	Pigment only	0
				Continuous late 0	Drusen	18
					Drusen + pigment	11
Group IV: thick continuous BLamD	Basal mounds only	20	11.3 ± 5.3	Early only 0	Normal	020	86 ± 5	0.796 ± 0.362	6/37.5; 20/125
					Patchy late 0	Pigment only
			Continuous late 20	Drusen	0
					Drusen + pigment	0
	Basal mounds + soft drusen	17	9.9 ± 2.1	Early only 0	Normal	0	81 ± 5	0.394 ± 0.337	6/15; 20/50
					Patchy late 0	Pigment only	0
				Continuous late 17	Drusen	0
					Drusen + pigment	17

 Clinical drusen defined as intermediate or large drusen.

*  From 2 patients. Eyes were normal at last clinical examination. Found to have soft drusen on histopathological examination 3 and 5 years later.

Table 4.

 

BLamD and Membranous Debris: a Summary of Clinical Correlations and Significance to AMD Pathogenesis

	Entity	Location	LM Appearance	EM Appearance	Clinical Correlation	Significance
BLamD	Early	Extracellular material between the RPE basal plasma membrane and its basement membrane.	Stains blue on picro-Mallory and shows faint vertical striations. May contain small unstained spaces. May be absent, patchy or continuous and reaches maximum height of half an RPE cell (7 μm).	Exists in three forms: fibrillar, amorphous (light and dark), and polymerized (banded). 6 Light, amorphous form is continuous with the RPE basement membrane. Polymerized form is similar to long-spacing collagen.	Compatible with normal fundus.	Presence of fibronectin, laminin and glycoproteins implies it is excess basement membrane material. 37 Considered Inert, but reflects escalating damage to RPE. Continuous early BLamD plus BLinD represents threshold early AMD.
	Late	A later development that lies above (internal) to the early form. Occurs in three stages: inclusions, nodules and continuous segments associated with increasing abnormality of overlying RPE.	Stains red on picro-Mallory. Patchy late BLamD begins as small, globular bodies (inclusions) in the order of 4 μm, lying within early BLamD, followed by nodular BLamD (nodular excrescences 14 lying beneath a single abnormal RPE cell. Continuous late BLamD, also known as “diffuse thickening of the Internal aspect of Bruch’s membrane,‘ 15 occurs as a confluent layer ≥250 μm in length and, together with the underlying early form, may be up to 25 μm thick.	Inclusions may indent RPE basement membrane. Later develops a more compact structure than early BLamD, with a wavelike appearance described as flocculent 9 or multilaminar.	Patchy late BLamD may occur in association with focal hyperpigmentation. Continuous late BLamD is always associated with clinical pigment changes.	When continuous, it is a sign of severe RPE dysfunction and incipient RPE atrophy.
Membranous debris	Dispersed	Membranovesicular material found in three locations: (1) between RPE basal plasma membrane and its basement membrane; (2) transiting through the RPE basement membrane; or (3) within the inner and outer collagenous zones of Bruch’s membrane.	Not seen.	Appear to bleb from basal surface of RPE cells. May be found between strands of early BLamD. Vesicles smaller and more compact when seen within RPE basement membrane. May have contained lipid removed by processing. 13	Not seen. Compatible with a normal fundus.	Early sign of RPE dysfunction. May result from nonapoptotic blebbing or dysregulated lipid trafficking by damaged RPE. 35 36 Fragments lying external to the RPE basement membrane contribute to age-related thickening of Bruch’s membrane 38 and may attract macrophages. 39
	BLInD	Occurring in conjunction with dispersed material, BLinD is a layer of membranous debris between the RPE basement membrane and the inner collagenous zone of Bruch’s membrane.	In semithin sections, appears as an unstained, narrow interval between the RPE and Bruch’s membrane.	A layer of membranous debris, thickest when adjacent to soft drusen.	Not seen. Often compatible with a normal fundus.	Forms a cleavage plane in which new vessels can spread. Represents threshold early AMD when continuous early BLamD is present.
	Basal mounds	Rounded collections of membranous debris between the RPE basal plasma membrane and its basement membrane, internal to BLamD.	Small, unstained spaces with rounded margins between attenuated RPE cells internally and early BLamD externally. A surrogate LM marker for the presence of BLinD.	Begin as pockets of membranous debris between two grossly abnormal RPE cells. The apices of the cells are stretched over the mounds but remain attached by their tight junctions.	Patients with basal mounds less likely to have normal fundus than those with BLinD alone. Large numbers of basal mounds correlates with poorer visual acuity. If over 30 μm in diameter, may account for dotlike drusen. 9	In the absence of soft drusen, mounds are found in older patients with pigment changes and poorer vision. Alternative and slower progression to advanced AMD compared with eyes with soft drusen.
	Soft drusen	Focal accumulation of BLinD between the RPE basement membrane and the inner collagenous zone of Bruch’s membrane.	May be all sizes, the smallest defined as at least half the height of an RPE cell. When regressing, the drusen appear granular and calcifications may develop.	Membranous contents which reduce as late BLamD is produced and membrane production ceases. Contains homogenous material 11 when in regression, probably corresponding to the granules seen on light microscopy.	Appear as intermediate or large drusen. When small, cannot be distinguished from hard drusen.	Occur in younger patients with better vision. Highest 5- and 10-year risk of advanced AMD, 20 21 in particular CNV. 22 23 24 25 Considered the hallmark of early AMD.

Figure 6.

 

Relation of basal deposits to AMD. Continuous early BLamD together with BLinD represent the threshold for onset of early AMD. The pathway then followed depends on the degree of membranous debris production. A large buildup manifests as intermediate or large drusen, whereas if limited to BLinD and basal mounds, the fundus initially may remain normal. At the same time, BLamD reflects the degree of RPE damage, with the clinical pigment abnormalities becoming apparent when late BLamD appears. Both pathways then progress toward geographic atrophy.