Download The Alabama Age-Related Macular Degeneration Grading

The Alabama Age-Related Macular Degeneration
Grading System for Donor Eyes
Christine A. Curcio,1'2 Nancy E. Medeiros,5 and C. Leigh Millican1
develop a systematic method for identifying and grading age-related macular degeneration (ARMD) in human donor eyes, postmortem fundus appearance was compared with
histopathologic assessment in eyes with a spectrum of age-related macular change.
PURPOSE. TO
METHODS. Eyes without grossly visible, late ARMD were obtained from 8 cancer patients and 26
donors older than 50 years. Postmortem fundus appearance was graded for drusen and pigmentary
change, using stereo color photographs. Eyes were processed and sectioned at 1 jam for histopathologic evaluation of macular retinal pigment epithelium-Bruch's membrane complex. The histologic
diagnosis was compared with gross fundus appearance, clinical ophthalmic histories (n — 25), and
clinical fundus photographs that were graded using the Wisconsin Age-related Maculopathy
Grading System (n = 5).
Ten eyes met histopathologic criteria for early ARMD. A similar proportion of eyes
(27%-32%) was identified as affected by ARMD by other published histopathologic criteria. By
choosing eyes with at least one druse larger than 125 jam in diameter or an area of pigmentclumping 500 /Ltm in diameter that was visible in the postmortem fundus, ARMD cases were
identified with 90% sensitivity and 95% specificity.
RESULTS.
CONCLUSIONS. The
Alabama ARMD Grading System permits rational and standardized use of donor
eyes in studies that are directed toward understanding the pathogenesis of ARMD. (Invest Opbthalmol VisSci. 1998;39:1085-1096)
ge-related macular degeneration (ARMD), a disorder of
the macular area involving the retinal pigment epithelium (RPE), Bruch's membrane, and photoreceptors, is
a major cause of vision loss in the elderly.' Current animal
models2'3 do not exhibit the complete range of pathologic
features of ARMD. There is a critical need for a well-designed
study of human eyes with ARMD, particularly those with early
stages of disease, to guide informed development of improved
animal models. Precise assignment of maculopathy status is of
paramount importance in case- control studies of human tissues. In eyes obtained from eye bank donors, maculopathy
status has been denned by available clinical history, histopathologic assessment, or both.4'5 In contrast, study patients in
current clinical epidemiologic ARMD research are characterized and staged by semiquantitative grading of standardized
fundus photographs.6'7 These long-term studies provide valuable information about ARMD natural history, including identification of fundus features indicating high risk for severe
disease.8"10 The process of relating morphologic, biochemical,
and histochemical findings in donor eyes to work in other
A
From the Departments of 'Ophthalmology and 2Physiologic Optics, University of Alabama at Birmingham; and 3Retina and Vitreous
Associates of Alabama, Huntsville.
Supported by grant EY06109 from the National- Institutes of
Health, Bethesda, Maryland; a grant from the Research to Prevent
Blindness, New York, New York (CAC); and a grant from the Helen
Keller Eye Research Foundation (NEM).
Submitted for publication July 24, 1997; revised November 14,
1997 and January 12, 1998; accepted February 11, 1998.
Proprietary interest category: N.
Reprint requests: Christine A. Curcio/Department of Ophthalmology, University of Alabama at Birmingham, AL 35294-0009.
disciplines would therefore benefit from information about the
fundus appearance of donor eyes used.
Because fundus photographs of donor eyes taken before
death are not routinely available, we sought a systematic
method for grading macular degeneration, using stereo color
photographs of the postmortem fundus. In this study, we
evaluated postmortem fundus appearance and RPE-Bruch's
membrane disease in well-preserved eyes with different degrees of age-related macular change. We validated fundus
grades by determining sensitivity and specificity for true ARMD
cases identified by histopathologic evaluation. We also calibrated the number of ARMD cases against the number identified by other ARMD standards, including published histopathologic criteria,11"14 clinical history, and grades for clinical
fundus photographs.715
Validation and calibration of a new ARMD grading system
are difficult because clinicians, epidemiologists, and pathologists use different definitions and even different names for
ARMD (for review, see Bird et al.15). There is reasonable agreement that the late form of ARMD includes changes that are
either exudative (RPE or neurosensory retinal detachments,
choroidal neovascular membranes with or without hemorrhages, exudates, and disciform scars) or nonexudative (geographic atrophy). 4121416 In contrast, early ARMD is variably
defined (Table 1), with the absence of late ARMD being the
only point of agreement. Histopathologic definitions of early
ARMD include the type or number of drusen and may include
basal deposits, RPE changes, or photoreceptor degeneration."" 14 Epidemiologic studies17"19 based on the Wisconsin
Age-related Maculopathy Grading System (WARMGS) for clinical fundus photographs717 include drusen type and changes in
RPE. The cross-disciplinary comparison in Table 1 arranges
Investigative Ophthalmology & Visual Science, June 1998, Vol. 39, No. 7
Copyright © Association for Research in Vision and Ophthalmology
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TABLE 1. Definitions for Early ARMD: Decision Criteria
Author/Study
14
Type*
Drusen
Logical
Ope\
Operator
Spraul et al.
Blue Mountain Eye Study19
H
E-C
Numerous
Soft, indistinct
AND
OR
Beaver Dam Eye Study17
E-C
Soft, indistinct
OR
Green and Enger12
International System15
Ramrattan et al.''
H
E-C
H
Soft
Soft
Numerous
AND
OR
RPE Changes
Grossly visible hypopigmentation or hyperpigmentation
Hypopigmentation or hyperpigmentation; soft distinct
drusen
Hypopigmentation or hyperpigmentation; hard distinct
drusen or worse
RPE or photoreceptor degeneration
Hypopigmentation or hyperpigmentation; drusen
None required
ARMD, age-related macular degeneration; RPE, retinal pigment epithelium.
"Type of study: H, histopathologic, using sections; E-C, epidemiology-clinical, using fundus photographs.
All definitions exclude late ARMD. Definitions are compared only for lesions visible in sections and in fundus. All histopathologic definitions
permit soft or numerous drusen or basal deposits.
early ARMD definitions in order from the most restrictive
(identifying the fewest cases) to the least restrictive (identifying the most cases). Definitions requiring that eyes meet two
criteria14 will identify fewer cases than definitions requiring
that eyes meet only one criterion.l' By similar logic, definitions
that include only a subset of soft drusen17 will identify fewer
cases than will definitions that include any soft drusen. 1215
Cases identified by different criteria are at slightly different
stages of disease, possibly underlying discrepant results across
studies." 14
To produce a histopathologic definition of early ARMD to
use as a reference standard for postmortem fundus grading, we
developed decision rules for histologic analysis logically similar
to those used for analysis of clinical fundus photographs. We
applied these rules to a group of quickly preserved donor and
surgically removed eyes with a spectrum of age-related macular
change. We then determined the best criteria for selecting
histologically confirmed ARMD eyes by postmortem fundus
appearance. Our overall goal was to define early ARMD in
terms of histopathologic features and fundus appearance, so
that as many of the same eyes as possible would be considered
ARMD by both methods.
METHODS
Our use of human tissues and clinical records was approved by
institutional review at the University of Alabama at Birmingham.
Tissue Collection and Photography
Our goal was to assemble a group of rapidly preserved eyes
with a spectrum of age-related macular change that would be
subjected to postmortem fundus photography and subsequent
histopathologic evaluation. To test the ability of the proposed
grading scheme to discriminate between eyes affected by
ARMD (ARMD eyes) and those without macular disease (nonARMD eyes), we sought similar numbers of age-matched eyes
from donors older than 50 years, with and without grossly
visible drusen and pigmentary change. A secondary goal was to
maximize the number of eyes with clinical histories.
Our results are based on the analysis of 34 eyes from 14
women and 20 men older than 50 years. Eyes were obtained
from 26 human donors and from 8 cancer patients, who had
undergone orbital exenteration for the removal of craniofacial
tumors. No donors or patients had diabetes. Median time to
preservation was 2 hours, 25 minutes (range, 1 hour 5 minutes
to 3 hours, 24 minutes) for donor eyes and 23 minutes (range,
6 to 40 minutes) for surgery eyes. The majority of eyes (n = 25)
came from a series of 57 consecutive donors or patients accessioned between August 1995 and April 1996. Maculopathy
status was unknown at the time of accession, except for three
donors who had histories of ARMD. During that period, each
surgical eye or one randomly chosen eye from each donor was
preserved for this study and underwent postmortem fundus
photography (see later description). Eyes from 24 donors in
the consecutive series were excluded from photography for
the following reasons: patient younger than 50 years (n = 8),
other grossly visible or clinically documented macular disease
(myopia, Roth's spots, exudates, central retinal vein occlusion;
n = 5), anterior segment surgery without clinical history (n =
3), late ARMD (geographic RPE atrophy and fibrovascular scars;
n = 2), normal eyes in age groups for which there were
enough specimens (n — 2), large folds in the fovea caused by
postmortem edema (n = 2), and patient too old for agematching (n = 1). Of the eyes of 33 donors and patients that
were photographed, 7 with gradable photographs were excluded from histopathologic evaluation because they were
grossly normal eyes without history or were in age groups for
which there were enough specimens (n = 5), or were damaged in processing (n = 2). Nine additional eyes from nonconsecutive donors and patients (December 1994-March 1997)
were photographed and sectioned to complete the study
group. These eyes had prominent drusen or pigmentary
change (n = 6) or were normal with clinical histories (n = 3).
Thus, eyes were excluded from the consecutive series and
nonconsecutive eyes were included to meet the overall goals
of the study design.
After removal of the cornea and lens, globes were fixed by
immersion for 24 to 48 hours in 0.1 M phosphate-buffered 1%
paraformaldehyde and 2.5% glutaraldehyde (n = 27), 4% paraformaldehyde and 0.5% glutaraldehyde (w = 5), or 4% paraformaldehyde (n = 2). Preserved globes were examined internally
after removal of superior and inferior calottes, anterior segment, and vitreous. At this time, we took stereo color photographs of posterior poles to specify early ARMD lesions. A
horizontal belt containing the macula was stabilized in a 5-ml
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Grading ARMD Donor Eyes
IOVS, June 1998, Vol. 39, No. 7
polystyrene cup and submerged in 0.1 M phosphate buffer to
reduce glare. We used a stereo microscope (model SMZ-U;
Nikon, Melville, NY) with a side port extension that allowed us
to produce stereo pairs and high-speed 35-mm film (Ektachrome EPJ320T; Eastman Kodak, Rochester, NY). We illuminated specimens in front of the retina with a fiber optic ring
light and behind the sclera with a dark-field base (epi-illumination and transillumination, respectively). Standard photographs
consisted of two stereo pairs at final magnifications on the slide
of X3 and X5.6 (see later description), respectively, both
taken with epi-illumination and transillumination at full power
(150 W). Supplementary photographs were taken with epiillumination or transillumination only to emphasize drusen or
pigmentary changes, respectively, in some eyes.
Clinical Histories and Fundus Photographs
Ophthalmic histories were obtained through eye bank-mediated letters to donor families and follow-up with eye doctors.
Histories were reviewed by a retina specialist (NEM) to exclude eyes with a history of chorioretinal disease that could
have been confused with early ARMD, such as high myopia,
epimacular proliferation, or other inherited retinal degeneration. In addition, we sought to compare our histopathologic
results with clinical findings. We emphasize that the histories
available for eye donors vary considerably in reliability and
validity. These records would be inadequate for clinical or
epidemiologic research, but they are typical of information that
is available for eye donors. As such, they are useful only for
determining a general level of visual function.
Photographs taken before surgery or death were obtained
for five eyes. To calibrate our results against current epidemiologic practice, these photographs were evaluated by five
graders experienced in applying the WARMGS to the Beaver
Dam Eye Study (BDES)17 or to the Age-Related Eye Disease
Study. Graders were unaware of patient history or histopathologic findings. Maculopathy status was assigned by the graders
using BDES criteria17 (Table 1). The same grades were used to
assign maculopathy status by criteria of the International System.15
Grading Postmortem Fundus Appearance
Stereo color photographs of the postmortem macula were
graded independently by two authors (CAC, NEM) before histologic analysis. We standardized the magnification of our
35-mm slides by photographing a ruler at different magnifications on the stereo microscope until 6 mm on the photograph
matched the diameter of the outer circle on a standard
WARMGS grid (nominally, 6000 jam on the retina). We created
acetate overlays with appropriately scaled grids and measuring
circles, using a software program (IntelliDraw; Aldus, Seattle,
WA). An overlay with WARMGS grid was centered and taped to
the left slide of the X 3 stereo pair. Donor or patient identity
was masked, and photographs were graded using a X4 viewer.
Tissue quality was assessed at three levels: 0, details of RPE
visible through a clear retina across the macula; 1, details
visible through the thin fovea and less visible through the thick
parafovea; and 2, details not visible in fovea or parafovea. Eyes
with grade 2 tissue quality were considered ungradable. Lesions were graded as absent, questionable, or present within
the grid. Lesions visible in the X5.6 stereo pair but not in the
X3 pair were considered questionable. Lesion size in the X3
1087
stereo pair was determined by fitting to circles 63 jam to 500
jam in diameter in a second overlay.15 The size of the largest
druse and predominant drusen were graded (<63 jam, 63-125
jam, and >125 jam). We also graded the total number (1-5,
6-10, 11-20, >20), the main and most central subfields, and
percentage of coverage by drusen in these subfields. With
regard to RPE changes, we graded the presence, location, and
size of hypopigmentation and hyperpigmentation. The latter
was defined as focal pigment clumps extending anteriorly into
the subretinal space or retina. For three or more discrete
pigment clumps, we graded the size of the bounding circle.
Histopathologic Assessment
The macular retina, RPE, choroid, and sclera were divided
horizontally with a razor, just superiorly to the foveal depression. The inferior half of each macula was subdivided into
blocks containing the fovea, temporal parafovea, and nasal
parafovea, which were osmicated, dehydrated, and embedded
in Epon-Araldite using standard procedures. The foveal and
temporal parafoveal blocks were serially sectioned at 1 jam
with a diamond Histo knife and stained with toluidine blue. We
examined two to three groups of serial sections that were at
least 60 /xm apart through the foveal rod-free zone. We used a
X60 objective, ocular reticules, and stepper motor stage with
digital readout of position.
Histopathologic assessment of the foveal and temporal
parafoveal blocks focused on lesions thought to be associated
with declining visual prognosis in ARMD. These lesions were
deposits in the inner aspect of Bruch's membrane/112'20 large
or confluent drusen,8'21 and focal hyperpigmentation of
RPE.810 We used published semiquantitative grading methods4'20 where possible (Table 2). Deposits internal to the basal
lamina of the RPE (basal laminar deposits)22 and those in the
inner collagenous zone of Bruch's membrane (basal linear
deposits)12 are indistinguishable in routine paraffin-embedded
sections.l2 Therefore, to identify cases in a manner comparable
to that used by other investigators, we did not distinguish
between them in our 1-jam sections, and we graded total basal
deposits, a combination of the two lesions (Table 2). We
measured the size of the largest druse and predominant drusen,
defined as focal deposits in the inner collagenous zone that
raised the RPE by half its typical height. In the case of confluent
drusen, size was measured for individual foci. For descriptive
purposes only, drusen shape and contents were noted. Although RPE changes such as clumping and atrophy are important sequelae of early-appearing basal deposits,l3 these changes
have not been included in previous grading schemes.4'20 We
graded the morphologic appearance of RPE (Table 2), treating
changes independent of the elevations associated with drusen.
Finally, we graded exudative lesions and changes in photoreceptor inner and outer segments.
Assignment of Maculopathy Status
Histopathologic assessments were graded for three purposes.
The first was for for case definition. Using the worst grades for
each parameter in the combined foveal and temporal sections,
we defined early ARMD as the presence of one large druse
(>125 jam in diameter; grade 2) or severe RPE changes (grades
2-4). Eyes with RPE changes also had to have at least one druse
(drusen number grade 1 or higher) or continuous basal deposits (grades 2-3). Note that the logical structure of this defini-
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IOVS, June 1998, Vol. 39, No. 7
TABLE 2. Histopathology Grades
Grades
Lesion
Total basal deposits
Size of largest druse
Number of drusen
RPE morphologic description
Detachment
Choroidal neovascularization
Photoreceptor outer segments
Photoreceptor inner segments
0
1
2
3
4
None
<66 ixm
None
Uniform
None
None
Normal
Normal
Patchy
63-125 jam
Thin continuous
>125 /mi
>3
Heaped or sloughed
Retina
Thick continuous
na
na
na
na
na
Anterior migration
Atrophy
na
na
na
na
na
na
na
na
1-3
Nonuniform
RPE
Present
Shortened
Broadened
na
Missing
Missing
na, not applicable; RPE, retinal pigmental epithelium.
tion resembles that used by epidemiologists for fundus grading
(Table 1) but differs in several significant ways. First, we used
drusen size as a stand-in for drusen type, because the histologic
equivalents of epidemiologic soft drusen subclasses are uncertain.13 Although most soft drusen are more than 63 jam in
diameter,13 we found in preliminary testing that using 63 /-ini
rather than 125 jLim as a size cutoff merely caused us to identify
more eyes without photoreceptor degeneration as ARMD
cases. Second, epidemiologists grading from stereoscopic
color fundus photographs require at least some drusen in eyes
with pigmentary changes to distinguish ARMD from other
conditions affecting the RPE (Table 1). We also included basal
deposits to improve discrimination among RPE lesions,13 because preliminary observations indicated that some of these
eyes had significant basal deposits without drusen.
The second purpose of the grading system was calibration
against existing case definitions. We used grading of histopathologic assessments to assign maculopathy status to the
same eyes, using other published definitions (Table i ) . 1 1 1 2 1 4
According to these studies, continuous basal deposits (grade
2-3), numerous drusen (grade 2), severe RPE change (grade
2-4), and photoreceptor degeneration (grade 1-2) meet criteria for the histologic diagnosis of early ARMD.
The third purpose of the grading system was validation of
postmortem fundus grades. Retinal edema and detachment can
appreciably reduce the visibility of posterior pigmented tissues. Our preliminary observations in fresh eyes suggested that
these changes had occurred after death and did not worsen
markedly with fixation. In addition, choroidal vessels empty
after death, making it difficult to assess minor pigmentary
variations in the plane of the RPE against the undulating background of pale vessels and dark interstitial tissue. Therefore,
applying methods for grading clinical fundus photographs directly to grading preserved donor eyes is unwarranted, given
the distinctly different detail visible in the fundus before and
after death. Our approach was to adapt WARMGS to the constraints of our material. To determine those constraints empirically, we compared fundus grades for the central subfield with
histopathologic assessment of the fovea and fundus grades for
the combined temporal inner and outer subfields with temporal histopathologic evaluation in eyes with gradable photographs. We also determined interobserver consistency. Using
histopathologic analysis as the standard, we determined the
number of true-positives and false-positives and true-negatives
and false-negatives for each grader's subfield scores. We considered the scoring of a grossly visible lesion by one grader a
false-positive for that grader if the lesion was not seen microscopically. However, we did not consider drusen that were
grossly visible to both graders but were not noted in histologic
study (n — 4) to be false-positive, because the tissue blocks
were not exhaustively sectioned, and it was possible to miss
lesions. Finally, we calculated sensitivity and specificity of
assigning maculopathy status on the basis of postmortem fundus appearance for histopathologically defined ARMD and nonARMD eyes using standard methods.23
RESULTS
Nineteen of 34 eyes were considered to have ARMD by at
least one standard (clinical history, histopathologic assessment, or postmortem fundus appearance). In Table 3, findings in 10 eyes that met our histopathologic criteria (cases
1-10) are summarized, plus findings in 9 eyes that met any
other criteria (A-I). Table 4 summarizes the number of
ARMD cases identified by different criteria, each normalized
by the number of eyes that could be evaluated and arranged
in order from most restrictive to least restrictive. Approximately one third (27%-33%) of eyes met either our histopathologic criteria, those of Green and Enger,l2 or those
of Spraul et al.14 to be classified as affected by early ARMD.
A similar proportion (33%) was identified by our fundusgrading criterion (see later description). In contrast, twice
as many eyes (46%-5O%) met either the Ramrattan et al.
criteria11 or had a history of ARMD or drusen. Within these
two broad groups, there was disagreement about individual
eyes (Table 3).
Clinical Findings and Fundus Appearance
Ophthalmic histories were available for 30 patients. The median time of the last ocular examination before death or exenteration was 11.3 months (range, 0-159.7 months). Thirteen
eyes (10 men and 3 women; 53-92 years old) had unremarkable maculae. Twelve eyes had clinical indication of ARMD or
drusen (Table 3; 7 men and 5 women; 60-86 years old): six
with drusen, two with changes in RPE, two with drusen and
changes in RPE, and two with nonspecific mention of ARMD.
A fluorescein angiogram in one eye (case 5) verified the absence of choroidal neovascularization. Clinical fundus photographs (Fig. 1) were evaluated forfiveeyes with ARMD- drusen
history. Three eyes (cases 2, 8, 5) contained mostly soft, distinct drusen, that is, solid, thick drusen of uniform density and
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ARMD Cases by Current Histopathologic Criteria
Other Criteria^
LE*
(mo)
Lens
Scored
PhotFA§
Age
Sex
60
63
64
M
F
M
0.0
9.7
na#
20/30
20/25+1
na
2
2
na
X/X/-
74
M
23.1
20/40-
3
X/-
74
M
18.7
20/20
2
X/X
74
M
0.0
20/50
3
X/-
75
80
M
M
0.6
0.3
20/60ph40
20/40-1
2
0
X/-
83
90
F
F
7
na
20/60
na
0
na
na
na
na
2
23.4
1.1
4.1
5.9
na
na
na
20/2520/60ph50+
20/100 +
20/40
20/50
na
na
na
2
0
3
2
3
12.9
20/40
OE
VAcc-f
Significant
Macular History
Drusen mild ARMD
Dry ARMD
No visual complaint
as per family
Drusen and/or
pigment changes
Dry ARMD, drusen,
pigment clumping
Prob ARMD,
pigment clump,
no drusen
Drusen
Few drusen, areas of
RPE atrophy
Giant drusen, ARMD
na
R
B
Y
Y
Y
Y
Y
Y
N
Y
N
Y
Y
Y
N
Y
N
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
N
N
Y
Y
Y
Y
N
N
Y
Y
N
Y
Figures
N
Y
Y
N
Y
ID, 2B, 6D
1A, 3, 6A
2A
Y
i c , 6C
Y
IE, 2E, 6E
Y
Y
6H
IB, 2D, 4B,6B
Y
Y
Y
Y
2C, 6G
2F, 4A, 6F
N
N
N
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
ses by Other Criteria
65
69
71
73
79
80
81
86
M
M
F
F
F
M
F
F
92
M
X
X
na
na
na
Drusen, dry ARMD
Drusen
Slight ARMD
Unremarkable
Few RPE changes,
dry ARMD
changes
Unremarkable
elated macular degeneration; FA, fluorescein angiogram; OE, orbital exenteration; PCIOL, posterior chamber intraocular lens; RPE, retinal pigment epithelial; Y, yes; N, no
ation before death or surgery,
y, corrected.
OL; cataract: 1, slight; 2, moderate; 3, significant.
us photos-fluorescein angiogram.
nd Enger12 (histologic examination); R, Ramrattan et al. 11 (histologic examination); S, Spraul and Grossniklaus.14 (histologic examination); B, Beaver Dam Eye Study17 (epi
onal criteria 15 (epidemiologic study); A, Alabama ARMD Grading System, postmortem fundus.
hy status of fellow eye: Hx, chart; A, Alabama ARMD Grading System, postmortem fundus.
le.
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TABLE
4. Early ARMD Cases Identified by Different Criteria
Criteria
Gradable
Eyes
Number of ARMD
Cases
Spraul et al.
Current (histologic)
Green and Enger
Current (fundus)
Clinical history
Ramrattan et al.
30
34
34
30
25
34
8
10
11
10
12
17
Percent
Affected
26.7
29.4
32.4
33.3
48
50
ARMD, age-related macular degeneration.
sharp edges with maximum size between 125 ju-m and 250 /xm.
A soft indistinct druse was identified in one eye (case 2) and
with less certainty in two other eyes (cases 8 and 5). The eye
in case 2 had a questionable drusenoid RPE detachment.13'21
Eyes in cases 2 (Fig. 1A) and 8 (Fig. IB) had increased pigment
within the central subneld. The eye in case 1 (Fig. ID) had only
soft distinct drusen without RPE changes. The eye in case 6,
which showed a distinctive coarse clump of increased pigment
and drusen (Fig. IE), was considered a pattern dystrophy.
Therefore, four eyes (cases 1, 2, 5, 8) met International criteria
for ARMD, and three met BDES criteria (cases 2, 5, 8).
Histopathologic Assessment
Ten eyes met our histologic criteria for early ARMD (cases
1-10; Table 3; Figs. 2, 3, 4). Eyes met criteria on the basis of
foveal disease alone, although ARMD was present in the temporal parafovea. As a group, these eyes had prominent basal
deposits (grades 2-3; n = 8; Figs. 2A, 2F), numerous drusen
(grade 2; n = 7), intermediate to large drusen (grades 1-2; n =
8; Figs. 2A, 2C; Fig. 3), RPE changes (grade 2-4; n = 6; Figs.
2C, 2D, 2E, 2F, 3B, 4B), and RPE detachments (n = 2). At least
mild photoreceptor degeneration (grade 1; inner or outer segments) was present in 9 of 10 eyes (Fig. 4). No eyes had
choroidal neovascularization or disciform scars. Seven eyes
with diverse changes in the foveal RPE-Bruch's membrane
complex, and unique combinations of lesions were agreed on
by all maculopathy standards to be early ARMD (Figs. 2, 3;
Table 2). The eye in case 2, with drusen present for at least 22
years before death, had a large druse with softening margins
located in the fovea24 (Fig. 3). Eyes in cases 4 and 7 had grade
2 basal deposits, grade 2 drusen size, and grade 2-3 RPE
changes (Fig. 2A). The eye in case 9 had RPE heaping and
atrophy, a serous RPE detachment, large calcified drusen, and
little or no basal deposits (Fig. 2C). The eye in case 8 had
macrophages in Bruch's membrane, intraretinal migration of
pigmented cells, and large drusen (Figs. 2D, 4B). Two eyes had
marked RPE involvement without detectable drusen. The eye
in case 10 had grade 3 basal deposits (Figs. 2F, 4A) and RPE
cells sloughed into the subretinal space (grade 2). The eye in
case 6 had a large mound of deposits containing abundant
pigmented debris and RPE heaping, migration, and atrophy
(Fig. 2E).
There was less agreement among standards about the
maculopathy status of eyes in cases 1 and 3 (Table 3)- These
eyes had large drusen, basal deposits, and mild photoreceptor
degeneration, but severe RPE change was absent (Fig. 2B).
There was also disagreement about the eye in case 5, which
had at least one large druse among numerous smaller ones.
Other eyes did not meet our criteria but met those of other
investigators (Table 3, donors A-I). Of note were four eyes with
clinical history of ARMD or drusen. Eye E had a few depigmented RPE cells that may have been interpreted as drusen.6
Eyes D and H had relatively mild ARMD in the temporal parafovea. Finally, eye F had a small area of RPE heaping and atrophy
without significant basal deposits, possibly related to a foveal
epiretinal membrane (H. Grossniklaus, personal communication, 1997).
Visual Acuity
To determine whether ARMD had markedly impaired vision
in these eyes, we compared histopathologic diagnoses with
best corrected visual acuity at last examination (Fig. 5).
Eight eyes with clinical ARMD- drusen history also met our
histopathologic criteria. Four eyes with ARMD history that
did not meet our criteria were considered non-ARMD and
are shown separately in the figure. Snellen acuity for 10
non-ARMD eyes and 6 of 8 ARMD eyes ranged from 20/20 to
20/40, within the range reported in large-population studies. 25 ' 26 Only five eyes had 20/50 vision or worse (three
non-ARMD, two ARMD). Poor acuity in three eyes with
ARMD-drusen history and mild pathology (Table 3: eyes E,
F, and H) was associated with other vision-limiting conditions. Eye E was last examined 1 day after cataract extraction, and eyes F and H had significant cataract. Of two ARMD
eyes with vision of 20/50 or worse, the one in case 6 had
significant cataract. Therefore, only in the eye in case 9,
with 20/60 vision through a posterior chamber intraocular
lens, can poor vision be ascribed primarily to ARMD.
Postmortem Fundus Appearance
Postmortem fundus appearance is shown for eight eyes with
early ARMD in Figure 6. Thirty of 34 eyes had gradable photographs. Four of the 26 donor eyes (15%) were ungradable
because of postmortem retinal changes. As a group, the more
rapidly preserved surgical eyes (Figs. 6B, 6D, 6E, 6H) had
clearer retinal tissue than did donor eyes (Figs. 6A, 6C, 6F, 6G)
but were nonetheless comparable to the best donor eyes.
Numerous drusen are present in most eyes with ARMD (Figs.
6A, 6B, 6C, 6D, 6G, 6H). Particularly distinctive were large
irregular drusen in the eye in case 2 (Fig. 6A) and calcified
drusen in the one in case 9 (Fig. 6G). Hyperpigmentation of the
RPE was present in many eyes (Figs. 6A, 6B, 6E, 6H), usually
associated with drusen. Two eyes had pigmentary changes
without obvious drusen. The eye in case 6 had a T-shaped
clump of hyperpigmented cells flanked by atrophic spots (Fig.
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FIGURE 1. Clinical fundus photographs. All eyes except (E) are considered affected by early age-related macular degeneration
(ARMD) according to international criteria because of the presence of soft drusen. The eyes in case 2 (A), case 8 (B), and case 5
(C) are considered affected by ARMD by the Beaver Dam Eye Study criteria because of the presence of increased pigment and soft,
indistinct drusen. (A) Case 2, 22 years before death, (B) Case 8, 1 week before orbital exenteration. (C) Case 5, 39 months before
death. (D) Case 1, 3 days before surgery. (E) Case 6, 1 day before surgery.
6E). The one in case 10 had fine pigment clumps (Fig. 6F).
Hypopigmentation of the RPE was present at the site of the
RPE detachment in the eye in case 9 (Fig. 6G).
For 30 eyes that were graded and then analyzed histologically, fundus grades were more reliable for eyes with
worse maculopathy than for eyes with no or borderline
maculopathy. Results for the fovea and temporal parafovea
were similar, despite the thicker parafoveal retina. No eyes
with large (>125 jam in diameter) or intermediate (63-125
ixm in diameter) drusen were missed by either grader.
Agreement on the size of the largest druse in these eyes was
excellent. However, the detection of small drusen (<63 /u,m)
generated false-negative and false-positive findings. Fine irregularities of foveal RPE pigmentation were occasionally
considered small drusen by one grader. Accordingly, agreement between graders was less good for measurements that
included small drusen (predominant drusen size, total
drusen number, and percentage of drusen coverage). We
also could not identify drusen subtypes except for calcified
drusen (Fig. 6G). No eye with severe RPE changes (histologic grades 2-4) was missed grossly, but several eyes with
normal RPE (grades 0-1) were graded as having small areas
of increased pigment by one or both graders. Rather than
missing subtle maculopathy, our errors tended in the direc-
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IOVS, June 1998, Vol. 39, No. 7
2. Histopathologic studies of eyes with early age-related macular degeneration.
One-micrometer sections; toluidine blue; scale bar in D, 20 fim. (A) Large druse with grade 2
basal laminar deposit (case 4). (B) Large druse (case 1). (C) Large calcified druse, retinal
pigment epithelium (RPE) atrophy, and photoreceptor loss (case 9). (D) Heaping and sloughing and anterior migration of the RPE, basal deposits, and macrophages in Bruch's membrane
{arrowhead; case 8). (E) Heaping and sloughing of the RPE with pigmented debris (arrowhead, case 6). (F) Grade 3 basal deposits (case 10).
FIGURE
tion of overinterpreting normal variation. Given these reliability constraints, we based our fundus criteria for early
ARMD on the size of the largest druse and area of increased
pigment and imposed a size criterion on the latter to reduce
the number of false-positive findings. By choosing eyes that
contained at least one druse more than 125 /xm in diameter
or an area of pigment clumping 500 jam in diameter within
the grid, we identified 9 of the 10 ARMD eyes, as well as 1
non-ARMD eye (Table 3), for a sensitivity of 0.90 and a
specificity of 0.95 (Table 5). Case 1 (Fig, 6D) was missed,
because its drusen were judged intermediate on gross inspection but were actually more than 125 /xm in diameter
when measured in sections. The non-ARMD eye (A, Table 3)
had large drusen in the outer temporal subneld that were
not sectioned.
DISCUSSION
To achieve the same goals of objectivity and reprodiicibility
that motivated clinical ARMD researchers to rely on standard
fundus photographs rather than clinical examinations,6'7 we
relied on standard photographs of the postmortem fundus
rather than on gross examinations for ARMD studies using
donor eyes. A photographic record of fundus appearance before irreversible processing of tissue permits retrospective examination of the macula in the light of subsequent results.
Although the gross lesions associated with late ARMD may be
more readily detectable in postmortem eyes,5'14 these eyes
represent advanced disease. Important contributions to our
current understanding of the critical early stages of ARMD have
been made by large-scale studies 41220 ' 27 " 29 and careful clinicopathologic correlations on a small number of eyes. 1316 ' 30
Further understanding of the cellular and molecular events
initiating ARMD will require biochemical and histochemical
analysis of donor eyes with well-defined maculopathy status.
Eyes with early ARMD can thus reveal useful information regarding ARMD pathogenesis, yet it is precisely this stage of
disease that is least agreed on. The Alabama ARMD Grading
System criteria are adequate to identify histopathologically
confirmed eyes with early ARMD with 90% sensitivity and 95%
specificity, thus permitting rational and standardized study
design. Our study eyes were chosen in a highly selective
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6
FIGURE 4. Photoreceptor degeneration in eyes with age-related macular degeneration. One-micrometer sections; toluidine blue; scale bar in B, 20 /xm. (A) Shortened and missing
outer segments and broadened inner segments (case 10). (B)
Missing photoreceptors (case 8).
3. Sections through the margin and center of a large
druse in the foveal center (case 2). One-micrometer sections;
toluidine blue; scale bar in C, 20 /jrnn. (A) Margin of large druse
contains pigmented debris, ribbons of basal deposits, and serous fluid, typical of softening drusen. (B) In a transitional zone
0.15 mm inferior to (A), pockets of original druse contents
appear (arrowheads). (C) Area 0.05 mm inferior to (B), center
of large druse that is representative of other drusen in this eye.
10
FIGURE
NonARMD
ARMD (clinical & histological)
ARMD (clinical only)
- 20/100
5 •
«
3^
2
manner, and therefore the proportions of ARMD and nonARMD eyes are not representative of prevalence in the general
donor population.
Because we found that most decreases in the clarity of the
retina and the visibility of underlying pigmented tissues occurred with death and not withfixation,we used eyes obtained
3 hours or less after death. Even among these quickly preserved eyes, however, 35% (2/57) of consecutive eyes were
not photographed because of folds in the macula, and 12%
(4/33) of eyes that were photographed could not be graded.
Nevertheless, our methods could be applied to a wider range
of postmortem intervals until fixation, provided that criteria for
tissue gradability are enforced. Interobserver reliability for definite maculopathy in postmortem eyes was good, despite some
confusion about small drusen and minor pigment variation,
similar to clinical fundus grading.7 We set a lower limit of 500
/am on the diameter of RPE hyperpigmentation, because
smaller abnormalities tended to be false-positive findings.
These errors are likely to decrease in the future as a result of
our histologic analysis, permitting us to reduce or eliminate the
size criterion, Because only three eyes in our series had confirmed atrophy, we could not assess reliability for its detection.
Definitions of ARMD have evolved -with little agreement
on the definition of specific lesions or classification.15'31 In
20/200
•
O
T
•
- 20/60
O
• O •d»
TO
1 •
T
•
- 20/40
•
•• o
- 20/20
ao
100
Age, yr
FIGURE 5-
Best corrected acuity (minimum angle of resolution
[MAR] and Snetlen's chart) at last examination for 30 donors or
patients. Eight patients had clinical history of age-related macular degeneration (ARMD) or macular drusen with histopathologic confirmation, 4 had clinical history without histopathologic confirmation, and 18 had history and histopathologic
confirmation of normal maculae. Pathologic analysis confirmed
that there was no optic nerve involvement in the patients who
underwent orbital exenteration. One patient had paracentral
visual field loss corresponding to a chronic occipitotemporal
lobe infarct revealed by magnetic resonance imaging. In two
patients, the last reliable visual acuity before the onset of tumor
complications (approximately 2 years before surgery) are plotted. Grades for lens status in eyes more than 50 years old (Table
3) for 8 ARMD patients (2/0/4/2), 4 clinical ARMD patients
(1/0/1/2), and 12 non-ARMD patients (0/2/5/3; 3 not recorded)
indicate a similar proportion of cataract in ARMD and nonARMD eyes and a higher proportion of significant lens changes
in eyes with clinical ARMD history only.
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IOVS, June 1998, Vol. 39, No. 7
6. Maculae in preserved eyes. Clinical fundus photographs for the eyes in (A), (B), (C), (D), (E) are shown in
Figure 2 (A, B, C, D, E), Photographs were taken with epi-illumination and transillumination at X5.6 original magnification, except for (A), which was taken with epi-illumination only. (A) Case 2; (B) case 8; (C) case 5; (D) case 1; (E) case
6; (F) case 10; (G) case 9; (H) case 7,
FIGURE
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IOVS, June 1998, Vol. 39, No. 7
TABLE 5. Comparison of Postmortem Fundus
Appearance and Histopathology
Histologically Defined
Postmortem
Fundus
True ARMD
True Non-ARMD
ARMD
Non-ARMD
Sensitivity/Specificity
9
1
0.90
1
19
0.95
ARMD, age-related macular degeneration.
calibrating our histopathologic ARMD criteria against other
standards, we confirmed the prediction that the number of
identified cases could range considerably. Nevertheless, the
percentage of eyes identified as cases by our criteria were in
good overall agreement with those identified by two other
studies.1214 The less restrictive logic of the Ramrattan et al.
criteria1' identified 50% more cases, mainly by including eyes
with numerous small drusen. Notably, the number of eyes with
clinical history of ARMD or drusen exceeded the number of
histopathologically confirmed ARMD cases by a similar margin.
These results underscore the fact, well-recognized in clinical
research, that medical records are insufficient to define cases.
Therefore, as in clinical ophthalmic research, ARMD cases
among donor eyes should be ascertained with independent
and objective measurements of maculopathy status, such as
histopathology, fundus appearance, or both. Despite these
limitations, however, the records we obtained were sufficient
for our original purposes—that is, to exclude non-ARMD chofioretinal macular pathology and to determine whether ARMD
pathology had seriously reduced vision. Corrected acuity at last
examination in 9 of 10 eyes with early ARMD were comparable
to those in age-matched non-ARMD eyes. The worst acuities
were associated with confounding factors such as cataract.
To our knowledge, this represents the first published
histopathologic study of eyes for which clinical photographs
were graded according to WARMGS. Offiveeyes with gradable
photographs, three were considered affected by ARMD by
BDES standards because of the presence of soft, indistinct
drusen, and in the case of two eyes, increased pigment. Soft,
indistinct drusen are thought to indicate high risk for late
ARMD.10 Their histologic equivalents are uncertain but are
probably caused by differences in drusen size, shape, RPE
depigmentation, and perhaps molecular constituents.32"35 A
druse type common to these three eyes had sloping margins
and membranous contents (preliminary observations).2427'36
Identification of high-risk drusen requires detailed grading and
serial section histologic analysis in the same eye. We confirmed
that focal hyperpigmentation corresponds to heaped,
sloughed, or migrating RPE cells.14 Of note, a distinctive pigmentary figure in the eye in case 6 was thought by WARMGS
graders to be a pattern dystrophy37"40 and therefore nonARMD. This finding underscores the fact that without clinical
examination and testing of donor family members, it is difficult
to distinguish between some hereditary macular. diseases37'41
and ARMD in donor eyes.
Despite the distinctly different appearances of the fundus
before and after death, we learned lessons that are potentially
applicable to clinical fundus grading. Clinical studies have
shown that the presence of any large drusen8 or any of a
1095
subtype of soft drusen,10 regardless of drusen number or degree of confluence, indicates an eye at risk for late ARMD. We
demonstrated the diversity of ARMD attainable by selecting for
only one large druse. In addition, we found that ARMD status
was established by foveal disease alone, consistent with clinical
studies showing that eyes with lesions less than 1500 jam from
the foveal center are at higher risk for late ARMD.42 It is
possible that future fundus grading could therefore be restricted to a more central area of the macula.
A single definition for early ARMD applicable to fundus
appearance and histology is desirable because it would ensure
comparable disease stage in case-control studies drawn from
different patient populations. As seen in even our small sample,
perfect agreement between the two is not attainable because
fundus photographs and histologic sections differ in how the
macula is sampled and in the precision with which lesions can
be measured. In addition to logical inconsistencies pointed out
in Table 1, it is also clear that histologic definitions of early
ARMD cannot be resolved with clinical definitions if they
include lesions not visible in the fundus. These lesions include
basal deposits,12'20 a prominent feature with a controversial
role in ARMD pathogenesis,43'44 and photoreceptor degeneration, the basis of ARMD-associated visual dysfunction. Other
investigators have shown that large drusen13 and RPE hyperpigmentation and hypopigmentation14 serve as markers for
basal deposits in the fundus. Our work demonstrates that large
drusen and changes in RPE may serve as markers for early
photoreceptor degeneration as well.
Acknowledgments
The authors thank Andy Patrick, Technical Services Director,
the Alabama Eye Bank, for timely retrieval of donor eyes; the
Montana Eye Bank for one pair of donor eyes; the Eye Foundation Hospital and the Tissue Procurement Program of the
UAB Comprehensive Cancer Center (CA13148) for surgical
specimens; Cynthia Owsley, of the Clinical Research Unit,
Department of Ophthalmology, University of Alabama at Birmingham (National Institutes of Health grant AG04212), for
clinical fundus photogniphs and eye examinations for cancer
patients; James Keffer for obtaining eye histories from donor
families; James O. Powell for consultation on eye disease;
Gerald McGwin, Jr., for consultation on data analysis (National
Institutes of Health AG04212); and Sarah Ansay, Jane Armstrong, Maria Swift, Jeff Whitehead, Larry Hubbard, and Ronald
Klein (EY06594), of the Fundus Photograph Reading Center,
Department of Ophthalmology and Visual Sciences, University
of Wisconsin, Madison, for evaluation of clinical fundus photographs.
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