Estimated Retinal Ganglion Cell Counts in GlaucomatousEyes with Localized Retinal Nerve Fiber Layer Defects

ANDREW J. TATHAM, ROBERT N. WEINREB, LINDA M. ZANGWILL, JEFFREY M. LIEBMANN,CHRISTOPHER A. GIRKIN, AND FELIPE A. MEDEIROS

� PURPOSE: To estimate retinal ganglion cell (RGC)losses associated with visible glaucomatous localizedretinal nerve fiber layer (RNFL) defects.� DESIGN: Observational cross-sectional study.� METHODS: A multicenter study of 198 normal eyes(138 subjects) and 66 glaucomatous eyes (55 subjects)recruited from the Diagnostic Innovations in GlaucomaStudy and the African Descent and Glaucoma EvaluationStudy. All eyes underwent standard automated perimetry(SAP), spectral-domain optical coherence tomography,and fundus stereophotography within 6 months. Glau-comatous eyes were included if localized RNFL defectswere detected by masked grading of stereophotographs.The number of RGCs in each sector of a structure-function map was estimated using a previously publishedmodel combining RGC estimates from SAP and spectral-domain optical coherence tomography. The estimatedpercentage loss of RGCs (combined structure-functionindex) was calculated.� RESULTS: In glaucomatous eyes, there were 136sectors with visible RNFL defects and 524 sectorswithout visible RNFL defects. The most common sectorswith visible RNFL defects were inferior and inferotempo-ral sectors, followed by superior and supertemporalsectors. Eyes with visible RNFL defects had a mean esti-mated RGC count of 657 172 cells versus 968 883 cellsin healthy eyes (P < .001). The average combinedstructure-function index in sectors with a visible RNFLdefect (59 ± 21%) was significantly higher than in sectorswithout a visible RNFL defect in glaucomatous eyes(15 ± 29%; P < .001) and higher than in healthy eyes(1 ± 13%; P < .001).� CONCLUSIONS: Although visible localized RNFLdefects often are considered an early sign of glaucoma,this study indicates that they are likely to be associatedwith large neuronal losses. (Am J Ophthalmol2013;156:578–587. � 2013 by Elsevier Inc. All rightsreserved.)

Accepted for publication Apr 8, 2013.From the Hamilton Glaucoma Center and Department of

Ophthalmology (A.J.T., R.N.W., L.M.Z., F.A.M.), University ofCalifornia, San Diego, La Jolla, California; the Department ofOphthalmology (J.M.L.), New York Eye and Ear Infirmary, New York,New York; and the Department of Ophthalmology (C.A.G.), Universityof Alabama, Birmingham, Alabama.

Inquiries to Felipe A. Medeiros, Hamilton Glaucoma Center,University of California, San Diego, 9500 Gilman Drive, La Jolla, CA92093-0946; e-mail: fmedeiros@glaucoma.ucsd.edu

578 � 2013 BY ELSEVIER INC.

THE HISTOLOGIC HALLMARK OF GLAUCOMA IS

accelerated loss of retinal ganglion cells (RGCs)and RGC axons.1–3 Loss of RGCs results in

thinning of the retinal nerve fiber layer (RNFL),4–6

topographic changes to the optic nerve head,1 and a reduc-tion in visual field sensitivity, which ultimately can resultin visual impairment and blindness.6,7

Glaucomatous damage to the optic nerve head andRNFL can be quantified using methods such as confocalscanning laser ophthalmoscopy, scanning laser polarim-etry,8 and optical coherence tomography (OCT).9–14

RNFL loss also can be observed directly by dilatedfundus examination using slit-lamp biomicroscopy andan accessory biconvex noncontact hand-held lens.15 Inhealthy eyes, the RNFL has a bright, uniform, striatedappearance and is most readily identifiable where theRNFL is thickest, close to the superior and inferior polesof the optic disc.5 The RNFL striations represent bundlesof RGC axons separated by Muller cell processes. Thebright component of a striation is the result of the highlyreflective parallel RGC axon bundles, and the dark bandsbetween are the result of the thick nonreflective dividingMuller cell glial septa.16

Loss of RGC axons leads to a reduction in the reflectivityof RGC axon bundles and may be diffuse or localized.17,18

Diffuse axonal loss results in a general reduction in RNFLstriations, a darkened appearance to the inner retina, andincreased visibility of the retinal blood vessels, whichnormally are embedded in the RNFL.5,16,18 There is someevidence that localized loss is less common but moreeasily visible because it has sharply demarcatedborders.15,19 Localized RNFL defects tend to be wedgeshaped and narrower as the RNFL bundles convergetoward the disc margin.20 Although localized RNFL defectsoccasionally are caused by nonglaucomatous optic neurop-athies, optic disc drusen, papilledema, or ischemic retinop-athies, they have a high specificity for glaucoma and are notfound in healthy eyes.20–22 It has been proposed thatlocalized RNFL defects may be the earliest observableclinical sign of glaucomatous damage.7,17 However,a previous primate histologic study found that a localizedRNFL defect was apparent on fundus photographs only ifmore than 50% of the thickness of the RNFL had beenlost.4 Similar findings were found in another histologicstudy of primate and human eyes in which localized lossof 28% to 45% of RNFL thickness was required for visibleRNFL defects.5 This suggests that rather than being an

0002-9394/$36.00http://dx.doi.org/10.1016/j.ajo.2013.04.015

ALL RIGHTS RESERVED.

early sign of glaucoma, a large number of RGCs are likely tohave been lost before a visible RNFL defect develops.

Although direct quantification of RGCs in vivo is notyet possible, empirical formulas derived from clinical struc-tural and functional tests may be used to estimate thenumber of RGCs. Estimates derived using these formulashave shown good correlation with histologic RGC countsin experimental glaucoma models.6 In recent studies, weproposed a method for estimating RGC loss from a combi-nation of standard automated perimetry (SAP) and RNFLassessment using OCT.23–25 The combined structure-function index (CSFI) was devised to represent thepercentage of RGCs that an eye has lost compared withthe expected number of RGCs. The combined structure-function index performed significantly better than isolatedstructural and functional parameters for staging the diseaseandmonitoring glaucomatous progression.23 The estimatednumber of RGCs and the combined structure-functionindex may also be calculated for individual regions of theoptic nerve and retina using a structure-function mapthat relates the location of RGC soma to the position ofRGC axons at the optic disc. The aim of this study wasto estimate the quantity of RGCs lost in regions associatedwith localized RNFL defects visible on stereophotographs.RGC estimates in sectors with an RNFL defect werecompared to RGC estimates in sectors without an RNFLdefect and to estimates in healthy eyes.

METHODS

THIS WAS AN OBSERVATIONAL, CROSS-SECTIONAL STUDY

involving participants from 2 prospective longitudinalstudies: the African Descent and Glaucoma EvaluationStudy and the Diagnostic Innovations in Glaucoma Study.Informed consent was obtained from all the participants,and the institutional review boards and human subjectscommittees of all 3 sites prospectively approved all of themethods. All methods adhered to the tenets of the Decla-ration of Helsinki for research involving human subjects,and the study was conducted in accordance with HealthInsurance Portability and Accountability Act regulations.The 3-site African Descent and Glaucoma EvaluationStudy collaboration includes the Hamilton GlaucomaCenter at the Department of Ophthalmology, Universityof California, San Diego (UCSD; data coordinatingcenter); the New York Eye and Ear Infirmary; and theDepartment of Ophthalmology, University of Alabama,Birmingham. The Diagnostic Innovations in GlaucomaStudy includes only patients recruited at UCSD and theprotocol is identical to that of the African Descent andGlaucoma Evaluation Study. Methodologic details havebeen described previously.26

The study included 66 eyes of 55 subjects with glaucomaand localized RNFL defects visible on fundus stereophoto-

VOL. 156, NO. 3 RETINAL GANGLION CELL LOSS AN

graphs and 198 eyes of 138 healthy subjects. Healthysubjects were recruited from the general population andhad intraocular pressure of 21 mm Hg or less with nohistory of raised intraocular pressure and normal SAPresults. At each visit, subjects underwent a comprehensiveophthalmologic examination including review of medicalhistory, visual acuity, slit-lamp biomicroscopy, intraocularpressure measurement, gonioscopy, dilated funduscopicexamination, stereoscopic optic disc photography, Spectra-lis spectral-domain (SD) OCT (Heidelberg Engineering,Dossenheim, Germany), and SAP using the Swedish inter-active threshold algorithm (SITA standard 24-2; Carl ZeissMeditec, Inc, Dublin, California, USA). Only subjectswith open angles on gonioscopy were included. Subjectswere excluded if they had a best-corrected visual acuityof less than 20/40; spherical refraction outside 6 5.0 diop-ters, cylinder correction outside 3.0 diopters, or both; orany other ocular or systemic disease that could affect theoptic nerve or the visual field.

� STEREOPHOTOGRAPH GRADING: All patients under-went simultaneous stereoscopic optic disc photography.Digital stereoscopic images were reviewed with a stereo-scopic viewer (Screen-VU stereoscope; PS Manufacturing,Portland, Oregon, USA) by 2 or more experienced graders.Each grader was masked to the subject’s identity and to theother test results. Details of the methodology used to gradeoptic disc photographs at the UCSD Optic Disc ReadingCenter have been provided elsewhere.26–28 Eyes withlocalized RNFL defects were identified by at least 2graders to be included in this analysis. RNFL defects weredefined as defects wider than twice the width of anarteriole, extending from close to the disc margin intothe parapapillary area, widening en route (i.e., wedgeshaped).20 Slit defects narrower than the diameter of adja-cent vessels were not included because they are foundfrequently in normal eyes.5,29

� IMAGING AND STANDARD AUTOMATED PERIMETRY:

The Spectralis SD OCT (software version 5.4.7.0) wasused to obtain RNFL thickness measurements. The deviceuses a dual-beam SD OCT and a confocal laser-scanningophthalmoscope that works by emitting a superluminescentdiode light with a center wavelength of 870 nm and aninfrared scan to provide images of ocular microstructuressimultaneously. A real-time eye tracking system is incorpo-rated that couples confocal laser scanning ophthalmoscopeand SD OCT scanners to adjust for eye movements and toensure that the same location of the retina is scanned overtime. A total of 1536 A-scan points were acquired froma 3.45-mm circle centered on the optic disc. All imageswere reviewed by the UCSD Imaging Data Evaluationand Analysis Center to ensure the scan was centered,that the signal strength was more than 15 dB, and thatthere were no artifacts. Scans that were inverted or clippedor those that showed coexistent retinal pathologic

579D NERVE FIBER LAYER DEFECTS

abnormalities were excluded. The RNFL segmentationalgorithm also was checked for errors.

All the patients underwent SAP testing using theSwedish interactive threshold algorithm standard 24-2strategy fewer than 6 months apart from imaging. All visualfields were evaluated by the UCSDVisual Field AssessmentCenter.30 Visual fields with more than 33% fixation lossesor false-negative errors or more than 15% false-positiveerrors were excluded. An abnormal SAP result was definedas having a pattern standard deviation outside the 95%confidence limits or a glaucoma hemifield test result outsidethe reference range. Data regarding optic disc area werecollected using the Heidelberg Retina Tomograph II(Heidelberg Engineering, Carlsbad, California, USA).27

� ESTIMATION OF RETINAL GANGLION CELL NUMBER:

The estimates of RGC counts were obtained according tothe model developed by Medeiros and associates based onempirical formulas derived by Harwerth and associatesfor estimating RGC counts from SAP and OCTresults.6,23–25 The details of the model and the empiricalformulas used to derive RGC counts have been describedin detail in previous publications.23–25 The initial step ofthe model consists of translating SAP sensitivity valuesinto RGC counts using formulas derived fromexperimental research in monkeys and subsequentlytranslated to human eyes. The following formulas wereused to estimate the number of RGC somas in retinalareas corresponding to specific SAP test field locations ateccentricity ec with sensitivity s in decibels:

m ¼ ½0:0543ðec31:32Þ� þ 0:9

b ¼ ½ � 1:53ðec31:32Þ� � 14:8

gc ¼ f½ðs� 1Þ � b�=mg þ 4:7

sector SAPrgc ¼ + 10 ðgc30:1Þ

In the above formulas, m and b represent the slope andintercept, respectively, of the linear function relatingganglion cell quantity (gc) in decibels to visual field sensi-tivity (s) in decibels at a given eccentricity. A structure-function map was used to assign SAP test locations to 10equal 36-degree sectors of the optic nerve head RNFL circlescan.6 Figure 1 shows the structure-function map for a righteye with sector 9 corresponding to the inferotemporalregion of the optic disc. In the left eye structure-functionmap, sector 9 also corresponds to the inferotemporal sectorof the optic disc, but numbering is counter clockwise. RGCestimates for individual SAP test locations were summed toestimate the number of RGC soma corresponding to eachregion of the optic disc.

The structural part of the model involved estimating thenumber of RGC axons from SD OCT RNFL thickness

580 AMERICAN JOURNAL OF

measurements. The model took into account the effect ofaging on axonal density and the effect of disease severityon the relationship between the neuronal and nonneuronalcomponents of the RNFL. The structure-function map had10 sectors, and therefore data from the commercially avail-able 6-sector RNFL thickness map was not used. Instead,mean RNFL thickness in each of the 10 disc sectors ofthe structure-function map was calculated from the 1536A-scan points of the 3.45-mm SD OCT circle scan (datafrom 154 adjacent A-scans were averaged for each ofsectors 1 through 6 and data from 153 adjacent A-scanswere averaged for sectors 7 through 10). The number ofRGC axons at each sector of the optic nerve was estimatedusing the following formulas:

d ¼ ð�0:0073ageÞ þ 1:4

c ¼ ð�0:263sMDÞ þ 0:12

sector OCTrgc ¼ 10 ðf½log10ðsector RNFL3108703dÞ�310� cg30:1Þ

In the above formulas, d is the axonal density (axons persquare micrometer), c is a correction factor for the severityof disease to take into account remodeling of RNFL axonaland nonaxonal composition, and sMD is the sector meandeviation. The sMD was calculated by measuring the devi-ation of the SAP sensitivity from the expected sensitivity ofSAP test locations in each sector.5 The expected valueswere derived from the Swedish interactive threshold algo-rithm normative database using the method described byHarwerth and associates (Table 1).6 The total SD OCT-derived RGC estimate was calculated as the sum ofOCTrgc in all 10 sectors. The estimates of RGC count ob-tained from SAP and SD OCT then were averaged, witha previously described weighting according to the overallseverity of disease, to obtain a weighted RGC (wrgc)count23,25:

sector wrgc ¼ �1þ sMD=30

�3sector OCTrgc

þ��sMD=30�3sector SAPrgc

The weights were chosen to reflect the inverse relation-ship of disease severity and SAP and SD OCT estimates,along the scale of MD values from 0 to �30 dB. The ratio-nale for the use of a weighting system has been described.25

Estimates of sector wrgc were summed to provide an overallwrgc estimate. A linear regression model then was used torelate wrgc estimates to age and optic disc area in healthyeyes and to develop a model to predict expected RGCnumbers. A model was generated for each sector of thestructure-function map using a development sample of169 healthy eyes recruited from the general population.To avoid model overfitting, none of the eyes in the devel-opment sample were included in the subsequent analysis.

SEPTEMBER 2013OPHTHALMOLOGY

FIGURE 1. Illustration of the structure-function map for the right eye used for sectorial estimation of retinal ganglion cell counts.(Left) The map divides the standard automated perimetry plot into 10 sectors, with 10 corresponding optic disc sectors, each of 36degrees. (Center) The location of the retinal nerve fiber layer defect was determined by fitting a 10-segment circle, corresponding tothe 10 zones of the structure-function map, to the optic disc photograph. (Right) In this example, the visible nerve fiber defect is insector 8.

TABLE 1. Predicted Mean Visual Field Sensitivities for Healthy Eyes in Each of the 10 Sectors of the Structure Function Map Used forSectorial Estimation of Retinal Ganglion Cell Numbers

Sector Number

1 2 3 4 5 6 7 8 9 10

Slope (dB/y) �0.057 �0.061 �0.060 �0.062 �0.057 �0.057 �0.061 �0.057 �0.062 �0.060

Intercept (dB) 34.68 33.60 33.32 31.68 31.68 31.68 31.19 33.46 33.01 34.45

After calculating the expected number of RGCs in eachsector for each eye, the sector CSFI was calculated as anestimate of the percentage of RGCs lost in each sector:

The global combined structure-function index wascalculated as the percentage of RGCs lost in the entireeye using the global wrgc. The location of the RNFL defectwas determined by fitting a 10-segment circle, correspond-ing to the 10 zones of the structure-function map, to theimage of the optic disc (Figure 1). The circumference ofthe 10-segment circle was adjusted for best possible fit tothe margin of the optic disc. The sectors corresponding

sector CSFI ¼ �ðexpected number of RGCs in sector� sector wrgcÞ=ðexpected number of RGCs in sectorÞ�3100

to the localized RNFL defects were determined by visualinspection (A.J.T.). If an RNFL defect straddled 2 sectors,it was deemed to involve both sectors.

� STATISTICAL ANALYSIS: Descriptive statistics includedmean and standard deviation for normally distributed vari-ables and median, first-quartile, and third-quartile values

VOL. 156, NO. 3 RETINAL GANGLION CELL LOSS AN

for nonnormally distributed variables. Student t testswere used for group comparison for normally distributedvariables, and Wilcoxon rank-sum tests were used forcontinuous nonnormal variables. For categorical variables,chi-square tests or Fisher exact tests were used.For glaucomatous eyes, the combined structure-function

index in sectors with localized RNFL defects was comparedwith the combined structure-function index in sectorswithout RNFL defects. The combined structure-functionindex in sectors with localized RNFL defects also wascompared with the combined structure-function index in

the healthy eyes. Clustering by participant and by eyewas included in the calculations to account for correlationsbetween eyes of the same subject and between sectors fromthe same eye. All statistical analyses were performed withcommercially available software (STATA, version 12;StataCorp LP, College Station, Texas, USA). The a level(type I error) was set at 0.05.

581D NERVE FIBER LAYER DEFECTS

TABLE 2. Comparison of Demographic Information, MeanDeviation on Standard Automated Perimetry, Mean Retinal

Nerve Fiber Layer Thickness, and EstimatedRetinal Ganglion

Cell Losses (Estimated Using the Combined StructureFunction Index) for Healthy and Glaucomatous Subjects

(Those with Visible Localized Retinal Nerve Fiber Layer

Defects; n ¼ 193 subjects, 264 eyes)

Healthy Eyes (n ¼ 198)

of Subjects (n ¼ 138)

Visible Localized RNFL

Defect (n ¼ 66 eyes,

n ¼ 55 Subjects) P Value

Age (y)a 62.5 (55.7 to 69.5) 63.4 (56.1 to 71.6) .248c

Race .001d

White 126 27

Black 72 39

Mean

deviation

(dB)

�0.1 (range, �1.0

to 1.0)

�3.3 (range, �5.9

to �1.4)

<.001c

Mean RNFL

thickness

(mm)

96.3 6 10.2 76.0 6 12.9 <.001b

Estimated no.

of RGCs

968 883 6 170 230 657 172 6 163 599 <.001b

Global CSFI

(%)

1 6 13 39 6 15 <.001b

CSFI1 (%) 4 6 19 10 6 27 .027b

CSFI2 (%) 1 6 21 13 6 31 .007b

CSFI3 (%) �2 6 22 30 6 22 <.001b

CSFI4 (%) �1 6 20 19 6 23 <.001b

CSFI5 (%) 2 6 28 16 6 30 .012b

CSFI6 (%) 3 6 27 4 6 34 .985b

CSFI7 (%) 2 6 23 26 6 28 <.001b

CSFI8 (%) �1 6 18 52 6 24 <.001b

CSFI9 (%) 2 6 21 52 6 26 <.001b

CSFI10 (%) 3 6 16 19 6 42 .004b

CSFI¼ combined structure function index; CSFI1 to CSFI10¼combined structure function index in each sector (1 to 10) of the

structure function map; dB ¼ decibels; RGCs ¼ retinal ganglion

cells; RNFL ¼ retinal nerve fiber layer.aMedian (interquartile range).bt test.cWilcoxon rank-sum test.dChi-square test.

RESULTS

THE STUDY INCLUDED 264 EYES OF 193 INDIVIDUALS. THERE

were 198 eyes of 138 healthy subjects and 66 eyes of 55subjects with localized RNFL defects visible on stereophoto-graphs. Seventy-six of 193 participants (39.4%) were ofAfrican ancestry, 113 of 193 participants (58.5%) were ofEuropean ancestry, and 4 of 193 (2.1%) participants wereof other racial background. Forty-nine of 66 eyes with local-ized RNFL defects (74.2%) had perimetric glaucoma, witha SAP defect present on 2 or more consecutive tests, and17 of 66 eyes (25.8%) had a localized RNFL defect, but norepeatable SAP defect. The demographic details and descrip-tive statistics are shown in Table 2.

Fifty-six eyes had a single RNFL defect, and 10 eyes hadRNFL defects at 2 discontiguous locations, resulting ina total of 76 RNFL defects. There were no eyes with local-ized RNFL defects at more than 2 discontiguous locations.Twenty-six contiguous RNFL defects (34.2%) involved 1sector, 40 contiguous RNFL defects (52.6%) involved 2sectors, and 10 contiguous RNFL defects (13.2%) involved3 sectors. Therefore, there were a total of 136 sectors withand 524 sectors without localized RNFL defects in the glau-comatous eyes. The most common sectors with localizedRNFL defects were sector 9 (52 eyes; 78.8%), sector 8(46 eyes; 69.7%), sector 2 (12 eyes; 18.2%), sector 3 (10eyes; 15.2%), sector 10 (7 eyes; 10.6%), and sector 7(6 eyes; 9.1%). Localized RNFL defects involved sector 1in 2 eyes and sector 5 in just 1 eye. There were no localizedRNFL defects in sectors 4 or 6.

Figure 2 shows the distribution of the combinedstructure-function index in sectors with visible RNFLdefects. In glaucomatous eyes, the combined structure-function index in sectors with visible RNFL defects(59 6 21%; range, 20% to 100%) was significantly higherthan in sectors without visible RNFL defects (15 6 29%;P < .001; Table 3). This indicates an average estimatedRGC loss of 59% in sectors with visible localized RNFLdefects, compared with an average estimated loss of 15%in sectors without visible RNFL defects. The mean CSFIin healthy eyes was 1 6 13%, which was significantly lessthan that in sectors without visible RNFL defects in glau-comatous eyes (P< .001). This suggests that there is signif-icant diffuse loss of RGCs in glaucomatous eyes, even insectors without visible RNFL defects. Eyes with localizedRNFL defects also had lower mean deviation (P < .001),RNFL thickness (P < .001), and estimated RGC count(P < .001) than healthy eyes (Table 2). The sector-combined structure-function indices in healthy eyes weresignificantly lower than in glaucomatous eyes (Table 2),with the exception of sector 6 (P¼ .985), a sector in whichthere were no RNFL defects. Figure 3 is a box plot showinglower quartile, median, upper quartile, and minimum andmaximum values of the combined structure-functionindex for all sectors of healthy eyes compared with the

582 AMERICAN JOURNAL OF

sector-combined structure-function index for sectors ofglaucomatous eyes with localized RNFL defects and sectorsof glaucomatous eyes without localized RNFL defects.The superior sector with the greatest number of RNFL

defects was sector 3. The mean estimated number ofRGCs in sector 3 was 57 475 6 20 186 (range, 5949 to78 964) cells in eyes with a localized RNFL defect in thissector compared with 137 559 6 33 080 (range, 70 131to 230 183) cells in sector 3 in healthy eyes (P < .001).The mean combined structure-function index in sector3 was 616 14% (range, 46% to 96%) in eyes with a local-ized RNFL defect in this sector compared with �2 6 22%(range, �72% to 51%) in healthy eyes (P < .001).

SEPTEMBER 2013OPHTHALMOLOGY

FIGURE 2. Histogram showing the distribution of estimatedretinal ganglion cell loss (estimated using the combinedstructure-function index [CSFI]) in sectors of glaucomatouseyeswithvisible localized retinal nerve fiber layer (RNFL)defects.

TABLE 3. Estimated Retinal Ganglion Cell Losses (EstimatedUsing the Combined Structure Function Index) by Sector for

Glaucomatous Eyes for Each of the 10 Sectors of a Structure

Function Map

Sector

No. of RNFL

Defects

CSFI for Sectors of

Glaucomatous Eyes

without RNFL

Defects (Mean 6

Standard Deviation)

CSFI for Sectors of

Glaucomatous Eyes

with RNFL Defects

(Mean 6 Standard

Deviation) P Valuea

1 2 9 6 27 43

2 12 5 6 27 50 6 17 <.001

3 10 25 6 18 61 6 14 <.001

4 0 19 6 23 No eyes with

defect in

sector 4

5 1 16 6 30 65

6 0 4 6 34 No eyes with

defect in

sector 6

7 6 23 6 27 62 6 18 <.001

8 46 39 6 22 58 6 22 .002

9 52 23 6 22 60 6 20 <.001

10 7 12 6 38 75 6 32 <.001

All 136 15 6 29 59 6 21 <.001

CSFI ¼ combined structure function index; RNFL ¼ retinal

nerve fiber layer.

Sectors with and without visible localized retinal nerve fiber

layer defects are compared (n ¼ 660 sectors).at test.

The inferior sector with the greatest number of RNFLdefects was sector 8. The mean estimated number ofRGCs in sector 8 was 65 965 6 34 931 (range, 1272 to133 074) cells in eyes with a localized RNFL defect inthis sector compared with 158 096 6 33 239 (range, 78085 to 259 719) cells in sector 8 in healthy eyes (P <.001). The mean combined structure-function index insector 8 was 58 6 22% (range, 20% to 99%) in eyes witha localized RNFL defect in this sector compared with �16 18% (range, �49% to 43%) in healthy eyes (P < .001).

Sectors with an RNFL defect had significantly highercombined structure-function indices than the immediatelyadjacent sectors (P< .001). The mean combined structure-function index in sectors immediately clockwise and coun-terclockwise to the sector involving the edge of the RNFLdefect was 226 24%. In eyes with localized RNFL defects,the sector with maximum combined structure-functionindex was sector 9 in 21 eyes (31.8%), sector 8 in 17 eyes(25.8%), sector 10 in 8 eyes (12.1%), and sector 3 in 8eyes (12.1%; Figure 4). The maximum combinedstructure-function index was in sectors 1, 2, and 7 in 3eyes each (4.6% each), sector 5 in 2 eyes (3.0%), and sector6 in 1 eye (1.5%). The mean combined structure-functionindex in the sector with the maximum combined structure-function index was 66 6 21%. The sector with themaximum estimated RGC loss was in the same sector asa localized RNFL defect in 50 eyes (75.8%) and in thesame or adjacent sector as a localized RNFL defect in 59eyes (89.4%). Examples of eyes with localized RNFLdefects are shown in Figures 5 and 6.

DISCUSSION

THIS STUDY DEMONSTRATES THAT LOCALIZED RNFL

defects visible on stereophotographs are likely to be associ-

VOL. 156, NO. 3 RETINAL GANGLION CELL LOSS AN

ated with loss of large numbers of RGCs. Sectors witha visible RNFL defect had an average estimated RGCloss of 59% (range, 20% to 100%) in the affected sector.This finding is in agreement with the results of histologicstudies that found focal RNFL defects are visible on fundusphotographs in regions where 28% to 50% of the thicknessof the RNFL had been lost.4,5 Localized RNFL defects,therefore, indicate substantial loss of RGCs.Sectors with an RNFL defect had significantly greater

estimated RGC losses than sectors without RNFL defectsand sectors of healthy eyes. For example, the superior sectorwith most RNFL defects was sector 3 (superior temporal).Eyes with an RNFL defect in sector 3 had an estimated80 000 fewer cells in the involved sector than healthyeyes, representing an estimated RGC loss of 58%. The infe-rior sector with most RNFL defects was sector 8 (inferiortemporal). Eyes with an RNFL defect in sector 8 had anestimated 92 000 fewer cells, which was also 58% lessthan estimated in healthy eyes. Examples of eyes withlocalized RNFL defects and large estimated RGC lossesare demonstrated in Figure 5 (Left column and Middlecolumn). The left eyes of 2 patients with visible inferotem-poral RNFL defects are shown. The RNFL defects eachinvolve 2 sectors with estimated RGC loss of 57% to68% and 81% to 85%. The SD OCT RNFL thickness

583D NERVE FIBER LAYER DEFECTS

FIGURE 3. Box-and-whisker plot showing the distribution ofestimated retinal ganglion cell loss (estimated using thecombined structure-function index) in all sectors of healthyeyes compared with the combined structure-function index insectors with visible localized retinal nerve fiber layer (RNFL)defects and the combined structure-function index in sectorsof glaucomatous eyes without visible localized RNFL defects.

FIGURE 4. Histogram showing the distribution of sectors withthe greatest estimated retinal ganglion cell loss (estimated usingthe combined structure-function index [CSFI]) for (Top)healthy eyes and (Bottom) glaucomatous eyes with a visiblelocalized retinal nerve fiber (RNFL) defect.

maps show inferotemporal thinning, and SAP shows supe-rior visual field defects, consistent with the location of thevisible RNFL defects.

Although most sectors with localized RNFL defects hadlarge estimated RGC losses, 11 (8.1%) of 136 sectors withan RNFL defect had estimated losses of less than 30%,including a single sector with an estimated loss of 20%.In all but 1 case, RNFL defects associated with an estimatedRGC loss of 30% or less also involved a neighboring sectorwith a greater estimated loss. An RNFL defect may havebeen visible in some sectors with a relatively low combinedstructure-function index because of the imperfect nature ofthe structure-function map. Also, localized RNFL defects,by definition, are areas of abnormality within areas of rela-tively preserved RNFL,15 and therefore, the visibility ofa localized RNFL defect depends on the thickness andarrangement of the RNFL in neighboring regions. MostRNFL defects were in the inferotemporal region followedby the supertemporal region. These regions are prone toglaucomatous damage, but also RNFL defects in these zonesare easier to detect because the RNFL is thicker.16–18,29,31

Figure 5 also shows an example of an eye with an RNFLdefect and relatively low estimated RGC losses (Rightcolumn). In this case, the sectors corresponding to thevisible RNFL defect had combined structure-functionindices of 21% and 35%; however, the adjacent sectorshad estimated RGC losses of only 0% and 5%. TheRNFL defect may be visible in this eye because of relativepreservation of RGCs in adjacent sectors.

In contrast, some sectors had no visible RNFL defectdespite high estimated RGC losses. Previous studies haveshown that eyes with advanced glaucoma are less likelyto have localized RNFL defects visible on stereophoto-graphs as defects broaden and become diffuse with disease

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progression.1,7,20,32 Therefore, there is no longer an areaof abnormality within an area of relatively preservedRNFL. Visibility of RNFL defects also depends on age,race, and disease severity.1,7,15,20 The RNFL is easier todiscern in younger subjects, and therefore focal defectsmay be detected more easily. Eyes of younger patientsalso have greater RGC counts. However, there was nosignificant difference in the age of subjects with glaucomaand healthy participants. In this study, there was a higherproportion of African Americans in the group withRNFL defects than in the group with healthy eyes. Thisis likely because RNFL defects are more visible in eyeswith more deeply pigmented retinal pigment epitheliumand choroid.Localized RNFL defects are considered one of the earliest

changes of glaucoma.7,17,32 In this study, 17 (25.8%) of 66eyes with visible RNFL defects had no repeatable SAPdefect, despite estimated global RGC losses of 25 6 9%.Figure 6 shows an eye with an RNFL defect involving 2 infe-rior sectors, but with SAP indices within statistically normallimits. Despite the normal SAP indices, the combinedstructure-function index indicates an estimated RGC lossof almost 50% in sectors corresponding to the visibleRNFL defect. These results indicate that RNFL defectsmay be associated with large RGC losses even in eyes withnormal visual fields. The spatial relationship between local-ized RNFL defects and the sector ofmaximal estimated RGCloss also was examined. The localized RNFL defects were inthe same or adjacent sector as the sector with the maximumcombined structure-function index in almost 90% of eyes.This suggests good correlation between the RGC estimatesand the stereoscopic photographs. There are, however, limi-tations to this comparison because the sector withmaximumRGC loss may be different from the sector with an RNFL

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FIGURE 5. Examples of 3 glaucomatous eyes with visible localized retinal nerve fiber layer (RNFL) defects. (Top row) The optic discphotographs show the estimated retinal ganglion cell loss (estimated using the combined structure-function index) in each of 10 sectorsof the structure-function map. (Top left) The first example shows a visible RNFL defect inferior-temporal to the optic disc. The esti-mated retinal ganglion cell loss in the 2 sectors corresponding to the defect is 57% to 68%. There is good agreement (Center left) withthe spectral-domain optical coherence tomography RNFL thickness map, which shows RNFL loss in the inferior-temporal region, and(Bottom left) with standard automated perimetry showing a superior arcuate defect. (Top center) The second example shows anRNFLdefect associated with an estimated retinal ganglion cell loss of 81% to 85%, and (Top right) the third example a RNFL defect asso-ciated with an estimated retinal ganglion cell loss of 21% to 35%. The corresponding (Center row) spectral-domain optical coherencetomography and (Bottom row) standard automated perimetry results for these eyes are also shown.

defect, for example, in an eye with advanced glaucoma anddiffuse damage in one hemifield and an RNFL defect in theother hemifield. Discordance between the location ofmaximum estimated RGC loss and an RNFL defect alsomay have been the result of individual anatomic variationsor the result of rotation of the OCT or disc photographduring image acquisition. Efforts were made to minimizerotation errors and to ensure correct alignment during imageacquisition. The Spectralis SD OCT also has a fovea-to-discalignment technology, which was used automatically totrack and anatomically to align circle scans and to help over-come measurement errors resulting from changing head oreye position during scanning.

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Other limitations of this study include the use of empir-ically derived formulas to estimate the number of RGCs.The original formulas were derived from studies in a primatemodel of glaucoma and were developed from studies usingtime-domain OCT.6 Despite these limitations, theformulas have been validated in multiple external humancohorts, including using SD OCT.23–25 There may havebeen eyes where localized RNFL defects were notdetected; however, efforts were made to minimize errorsby using 2 masked graders. RNFL defects might havebeen detected in more eyes and their borders might havebeen delineated better if we had used a different methodof photographic acquisition, for example, a red-free source

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FIGURE 6. (Left) Example of an eye of a 70-year-old subject with a localized retinal nerve fiber (RNFL) defect visible on optic discphotography. The RNFL defect corresponded to two sectors of the structure-function map with an estimated retinal ganglion cell lossof 42 to 48%. The estimated retinal ganglion cell loss in the other sectors is also shown. (Top right and Bottom center) Spectral-domain optical coherence tomography revealed localized thinning of the RNFL in the inferior-temporal region. (Bottom right)Despite high estimated retinal ganglion cell losses, standard automated perimetry global indices were within statistically normal limits.

of illumination. Red-free light is absorbed by melanin inthe RPE and choroid, and therefore creates a dark back-ground in contrast to the white striations of the RNFL.15

A computerized method of RNFL defect detection alsomight have identified localized RNFL defects in othereyes.33 We decided to use plain light stereophotographsbecause this reflects a commonly used method of clinicalassessment.

In conclusion, this study indicates that eyes with local-ized RNFL defects visible on stereophotographs are likelyto have, on average, 39% fewer RGCs than a healthy eye

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of a similarly aged subject. Localized RGC loss in the regionof the RNFL defect is likely to be particularly high, becausethe sector corresponding to the RNFL defect had onaverage 59% fewer RGCs than expected. Therefore, ratherthan being an early sign of glaucoma, localized RNFLdefects visible on stereophotographs and with slit-lampbiomicroscopy are likely to be associated with largeneuronal losses. The results suggest that sectorial analysisusing the combined structure-function index is a usefulmethod to identify areas of localized RNFL loss and glau-comatous damage.

ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTand the following were reported. Dr Medeiros has a patent pending on the combined structure and function index. Drs Weinreb, Zangwill, Liebmann,Girkin, and Medeiros receive research support from Carl-Zeiss Meditec; Drs Weinreb, Zangwill, Liebmann, and Medeiros receive research supportfrom Heidelberg Engineering; Dr Weinreb receives resource support from Optovue, Kowa, Nidek, and Topcon; and Dr Weinreb is a consultant forCarl-Zeiss Meditec, Inc, and Topcon. Supported in part by Grants EY021818 (F.A.M.), EY11008 (L.M.Z.), EY14267 (L.M.Z.), and EY019869(L.M.Z.) from the National Eye Institute, National Institutes of Health, Bethesda, Maryland; an unrestricted grant from Research to Prevent Blindness,Inc, New York, New York; and grants for participants’ glaucoma medications from Alcon, Allergan, Pfizer, Merck, and Santen. Involved in Design andconduct of study (A.J.T., R.N.W., L.M.Z., J.M.L., C.A.G., F.A.M.); Collection, management, analysis, and interpretation of data (A.J.T., R.N.W., L.M.Z.,J.M.L., C.A.G., F.A.M.); and Preparation, review, or approval of manuscript (A.J.T., R.N.W., L.M.Z., J.M.L., C.A.G., F.A.M.). Registration information isavailable at the National Institute of Health database at http://www.clinicaltrials.gov (identifiers, NCT00221923 and NCT00221897; September 14,2005).

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19. Tuulonen A, Airaksinen PJ. Initial glaucomatous optic diskand retinal nerve fiber layer abnormalities and their progres-sion. Am J Ophthalmol 1991;111(4):485–490.

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Biosketch

Andrew J. Tatham is Visiting Glaucoma Fellow at theHamiltonGlaucomaCenter, University of California, San Diego. He

undertook resident and glaucoma fellowship training at University Hospitals Leicester andMoorfields Eye Hospital, United

Kingdom. He has achieved several prizes including the Pfizer Ophthalmic Fellowship from the Royal College of

Ophthalmologists and the award for highest score at the European Board of Ophthalmology examinations. His research

interests are glaucoma imaging and structure-function relationships in glaucoma.

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