glaucoma remains the third or fourth most common cause of blindness in different regions around the globe .
since this is a treatable disease , early diagnosis and adequate follow - up are gaining importance .
also , direct and indirect economic burdens tend to increase for governments with patients extended lifetime expectancy and complexity of disease stage .
so far standard automated perimetry ( sap ) is still considered the  gold standard  method for function analysis even though defects are only detectable after substantial cell loss . since its introduction
, optical coherence tomography ( oct ) has turned into a fundamental tool in the evaluation of a variety of different retinal diseases , in particular primary open angle glaucoma ( poag ) .
thinning of the peripapillary retinal nerve fiber layer ( rnfl ) and full macular thickness ( mt ) have been largely used in poag evaluation [ 3 , 4 ] .
in addition , recent improvements in oct technology ( i.e. , spectral oct and software analysis ) not only increased image resolution but also allowed customized analysis of the individual retinal layers .
glaucoma damage is primarily related to the ganglion cells . thus oct retinal layer segmentation allows us to directly analyze the ganglion cell layer separately and thereby look directly at the site of damage . indeed ,
ganglion cell - inner plexiform layer ( gcipl ) segmentation can identify changes and correctly diagnose glaucoma with a similar sensitivity as the rnfl or optic nerve head ( onh ) parameters [ 68 ] .
thus segmentation may allow a more sensitive structure - function correlation in different stages of disease .
although the increased number of oct manufacturing companies may contribute positively to price competition as well as hardware and software improvements , it also leads to variability in measurements and analysis methodology . in this study
, we determined if the brand of the spectral - domain oct used and their respective segmentation programs influence structure - function analysis differently . to the best of our knowledge ,
this is the first study to compare two different oct systems and their respective macula layer segmentation software and associate them with sap in structure - function analysis .
the study protocol was approved by the ethics committee of the university of basel , and informed consent was obtained from all participants before the examination . all procedures followed the tenets of the declaration of helsinki .
the inclusion criteria included a visual acuity of 0.8 or better , and a refractive error between 6 diopters of hyperopia or myopia .
all glaucoma patients had a cup - to - disc ratio of at least 0.5 and a localized thinning of the neuroretinal rim on oct ( cirrus ) corresponding to the fundus examination .
the oct thinning should have at least one red sector or two yellow sectors on the thickness map ( less than 1% and 5% of the normal population , resp .
preperimetric glaucoma was defined by the presence of optic nerve abnormalities consistent with glaucoma and a normal visual field as tested with sap .
other glaucoma patients had to present a reproducible glaucomatous visual field defect on at least three examinations with a mean defect ( md ) higher than 2.0  db and/or a squared root of loss variance ( slv ) over 2.5  db .
individuals with previous ocular surgery , systemic diseases , or regular use of medications that could influence the eye ( e.g. , antidepressant , chloroquine ) were excluded from the study .
the right eye was included in the study , if it did not fulfill any exclusion criteria .
when not possible , visual field examination was performed at a maximum interval of 7 months from oct examination .
technical details from the two different commercially available oct instruments used are displayed in table 1 .
the pupil of the study eye was dilated with a solution of tropicamide 0.5% and phenylephrine 1% ( spital - pharmazie usb , switzerland ) before examination .
oct images were obtained in cirrus using the fast macular cube protocol 512  218 ( 128 horizontal scan lines each composed of 512 a - scans , cirrus sd - oct , carl zeiss , usa ) , and the fast volume scan in spectralis hra + oct ( 25 section scans and 26 art frames , heidelberg engineering , inc . , heidelberg , germany ) .
both instruments have a scan area of 6  6  mm and macular retinal thickness is calculated in microns in an area correspondent to the early treatment diabetic retinopathy study ( etdrs ) grid .
the mt values used in this study corresponded to the 1 and 3  mm circles of the etdrs grid .
gcipl thickness is calculated by cirrus software in the area of an elliptical annulus with a 2.0  mm vertical and 2.4  mm horizontal radius , excluding a central elliptical area ( 0.5  mm vertical and 0.6  mm horizontal radius ) that corresponded to the foveola . according to studies of human retina , the highest density of ganglion cells occurs in this area . as spectralis software ( version 6.0.3 ) uses the etdrs grid also for gcipl thickness calculation , values in the 3  mm circle were averaged , excluding the 1  mm circle , and compared to cirrus ( figure 1 , top ) .
cirrus software excluded the macular rnfl layer from the gcipl analysis while spectralis software calculated each retina layer separately ( figure 1 , bottom ) .
therefore , in spectralis , only the layers of interest in this study ( ganglion cell and inner plexiform layers ) were added in a separate microsoft excel spreadsheet .
the exclusion of rnfl in cirrus was based on the histologic observation that the macular gcipl layer presents less variation than the rnfl among normal individuals .
differences between the octs are that while both allow for manual corrections of the macular thickness boundaries , only spectralis allows for manual correction of possible errors in gcipl segmentation .
cirrus , but not spectralis , separately analyses the minimum value of gcipl thickness ( mgcipl ) measured within the areas analyzed .
thus parameters included in this analysis were averaged mt and the gcipl from both octs , and in addition , their average after manual correction ( cmt and cgcipl ) in spectralis and the mgcipl value given in cirrus .
all images included in this study had signal strength over 7 for cirrus and a quality score over 25 for spectralis ( limits considered as good / acceptable image quality for analysis , according to each instrument 's manual ) .
standard automated perimetry was performed using an octopus perimeter ( octopus 101 , g2 program , haag - streit ag , switzerland ) .
total field md ( mean defect ) values in db were included in the analysis .
all sap exams used in this study were inside reliability parameters ( fixation loss < 33% , false - positive and false - negative rates < 25% ) .
differentiation between glaucoma and controls within each measurement was assessed with a t - test and p values posteriorly adjusted with fdr ( false discovery rate ) .
the predictive diagnostic performance for each parameter , that is , the ability to differentiate glaucoma from control , was assessed calculating the receiver operating characteristics ( roc ) curves and the respective area under the roc curve ( auc ) .
a perfect predictive performance is represented by an auc of 1.0 which means that this parameter can differentiate glaucoma from control with 100% sensitivity and specificity , while an auc value of 0.5 means a prediction mostly influenced by chance .
aucs from different octs were compared with the delong test . for prediction of structure - function relationship
all calculations were adjusted to age . as oct values are linear values but sap are reported in db , a logarithmic value , oct values were transformed to logarithmic scale for better comparisons with sap .
results were expressed as the regressive slope coefficients ( on log - scale ) with corresponding standard errors and p values .
statistical analyses were performed using spss ( ibm spss statistics , version 22 ) , and the statistical package r   ( version 3.0.2 ) . in this study ,
the mean age was 59.5 years ( sd  13.9 ) for the glaucoma group and 49.2 years ( sd  7.0 ) for the controls ( p = 0.013 ) .
median md was 2.2 ( range : 0.417.0 )  db in poag ( including ppg ) and 0.2 ( range : 3.82.0 )  db in controls ( p = 0.024 ) . 

table 3 shows averages from mt and gcipl in both octs in the poag and control groups .
spectralis and cirrus showed a significant difference between patients and controls in both mt ( p = 0.018 , p = 0.028 , resp . ) and gcipl ( p < 0.001 , both ) .
manual correction of the software segmentation parameters in spectralis produced a significant difference between measurements in gcipl ( p < 0.05 ) but not in mt ( p = 0.715 ) .
a total of 2 controls ( 12.5% ) and 7 patients ( 41.1% ) needed posterior manual correction of retina thickness segmentation .
bland - altman analysis showed disagreement between octs in mt and gcipl values ( figure 2(b ) ) . on average , measurements with spectralis were thicker than with cirrus .
for mt the difference was 21.64  m ( sd  4.5 ) before and 21.65  m ( sd  4.5 ) after manual correction ( p < 0.001 ) . for average gcipl thickness
the difference was 9.8  m ( sd  5.4 ) before and 10.0  m ( sd  5.3 ) after manual correction ( p < 0.001 ) . with higher values measurements obtained with spectralis tended to differ more from those measured with cirrus .
this difference increased when we compared spectralis averages before ( 14.1  m , sd  5.9 ) and after correction ( 14.4  m , sd  5.8 ) with mgcipl ( p < 0.001 ) .
there was no significant difference between the age - adjusted aucs from mt in cirrus ( 0.798 ) and spectralis , before ( 0.801 ) and after ( 0.805 ) manual correction .
this was also observed between octs for gcipl measurements : 0.879 in cirrus and 0.886 before and 0.886 after correction in spectralis . minimum gcipl value in cirrus had an auc of 0.930 ( table 3 ; figure 3 ) .
mt and gcipl had a negative significant association with md ( p < 0.001 ) , in both poag ( p < 0.001 ) and controls ( p < 0.001 ) for cirrus and spectralis ( table 4 , figure 4 ) .
the aim of this study was to compare thickness measurements between two commercially available octs using their respective segmentation programs and assess if the brand of spectral - domain oct used might influence structure function analysis differently in glaucoma . using two different spectral - domain octs , cirrus and spectralis
, we observed that there is a significant difference in full macula and gcipl thickness measurements between machines .
therefore measurements are not interchangeable . nevertheless , when assessing structure function relationship individually , all measurements from both machines demonstrated a statistically significant relationship with function measured by standard automated perimetry .
further , age - adjusted aucs demonstrated that all measurements had a similar predictive performance and could correctly differentiate patients from controls .
a literature review in pubmed using specific terms ( optical coherence tomography , glaucoma , ganglion cell , macula , thickness , and segmentation software ) did not reveal any other study which we could directly compare to this one . while we compared the entire area within the central 3  mm diameters , most studies either refer to the central 1  mm area [ 1116 ] or compare the areas within the etdrs sectors calculated by the respective octs ( i.e. , the superior , inferior , temporal , and nasal sectors ) [ 11 , 16 ] . here
this is in accordance with a study from mylonas et al . , where spectralis macula thickness also showed the highest values , in the central retinal thickness ( crt , 1  mm diameter ) and individual sectors of the 3  mm area , in comparison to other octs ( including cirrus ) .
though the study was conducted in neovascular age - related macular degeneration patients ( 28 individuals ) , its control group ( 10 individuals ) showed the same pattern .
other studies found the same difference between cirrus and spectralis in crt [ 12 , 14 , 15 ] .
the gcipl average in early glaucoma patients from these studies ( 69.7  m ) is comparable to our study average ( 68.5  m ) .
a comparison of layer segmentation reproducibility was conducted at the iowa university using cirrus and their own segmentation software . here
, the overall average from cirrus gcipl was reported as 70.0  m ( sd  11.4 ) in glaucoma , which also did not differ much from the patient group average in our study ( cirrus ) .
recently , martinez - de - la - casa et al . demonstrated that , using spectralis layer segmentation software , macular rnfl thickness was the only parameter to differentiate healthy subjects from glaucoma suspects . we have not found a study comparing spectralis and cirrus segmentation software , most probably because spectralis software was made commercially available only recently . the clear difference in macula thickness between octs could be explained analyzing the specific retina boundaries established by each manufacturer . while the inner boundary is always the vitreoretinal interface , the outer retinal boundary varies between manufacturers . for cirrus
the outer boundary corresponds to the level of the interdigitations of the external layers of the photoreceptors in the retinal pigment epithelium ( verhoeff 's membrane ) , while in spectralis it is at bruch membrane [ 11 , 13 , 15 ] .
nevertheless , the establishment of different boundaries for total macula thickness calculation can not explain the significant difference between gcipl thicknesses we found in this study .
different image resolution , intrinsic reflectance , and analysis algorithms within each software may influence this calculation .
we also can not exclude an influence from the different areas analyzed , that is , ellipsoid in cirrus versus the annular in spectralis .
however both areas differ only slightly and include the highest density area for ganglion cells .
while total macula thickness boundaries can be manually corrected in both cirrus and spectralis , individual layer segmentation correction is possible only in spectralis .
we did not observe a significant difference in total macula averages before and after manual correction of inner and outer retinal boundaries in spectralis .
this could be explained by the observation that , specifically for the internal limiting membrane and bruch 's membrane , delineation errors occurred mostly in the extreme periphery of the image slice .
however , for the gcipl segmentation errors also occur within the 3  mm ring averages .
thus gcipl layer segmentation corrections made in spectralis resulted in significantly different values , while values remained significantly higher than with cirrus .
in addition , the same difference in thickness measure will impact less on the thicker total macula thickness but more on the thinner gcipl thickness .
. a significant correlation between function ( global md ) and morphology ( mt [ 23 , 24 ] or gcipl [ 20 , 25 , 26 ] ) has been demonstrated previously . in agreement , despite the significant difference between cirrus and spectralis measurements , both octs demonstrated a significant positive association with global md .
we are not aware of studies directly comparing the diagnostic performance between mt and gcipl in glaucoma . when compared to rnfl , mt had an inferior diagnostic performance in cirrus   and in stratus [ 3 , 23 ] . in our study ,
even though we found no other studies directly comparing auc between mt and gcipl , the auc values found in this study are in accordance with findings from other studies using mt   and gcipl [ 7 , 8 , 17 , 20 , 28 ] .
though we found a significant difference between oct measurements , the small population analyzed here may limit our findings . also , stage of disease might influence results given that the relationship between structural and functional damage is still not completely understood . including
in addition , knowing that age may influence our results , we adjusted all calculations for age .
difference in gender distribution is also a concern : cohn et al . did not observe a significant difference between males and females when comparing total db from sap .
total macular thickness is about 7.5  m thicker in men than in women . while age and sex differences should be considered when performing disease diagnostics , this is not the main scope of this study as we aimed to analyze measurements from two different octs within the same subject .
once this is incorporated , comparison between deviation maps from these octs could contribute to understanding differences between calculations .
finally , the spectralis segmentation software used here is a beta version . a definite version , without many changes ,
the significant difference between measurements from cirrus and spectralis octs does not allow free interchange of machines , for instance , in the follow - up of patients . in a clinical setting
, clinicians must be aware that once you change the machine and software analysis , a new baseline for the patient is needed .
nevertheless both machines showed similar capability of diagnostic performance in early glaucoma and also in their correlation to functional changes such as standard automated perimetry .
 
 purpose . to compare two different spectral - domain optical coherence tomography ( oct ) systems in regard to full macular thickness ( mt ) and ganglion cell layer - inner plexiform layer ( gcipl ) measures and in regard to structure - function correlation when compared to standard automated perimetry ( sap ) . methods . 
 seventeen primary open angle glaucoma patients and 16 controls ( one eye per subject ) were enrolled . 
 mt and gcipl thicknesses were measured by cirrus and spectralis octs . 
 octopus perimeter 101 ( g2 protocol ) reports sensitivity in mean defect ( db ) . 
 differences between measurements were assessed with student 's t - test and bland altman . 
 diagnostic performance was also compared between each parameter calculating the areas under the operator receiver ( roc ) . 
 linear models were used to investigate structure - function association between oct and sap . 
 results . disagreement between octs in both mt and gcipl values was significant . 
 spectralis values were thicker than cirrus . 
 average difference between octs was 21.64  m ( sd 4.5 ) for mt and 9.8  m ( sd 5.4 ) for gcipl ( p < 0.001 ) . 
 patients differed significantly from controls in both octs , in both measurements . 
 mt and gcipl were negatively associated with md ( p < 0.001 ) . 
 conclusions . although oct values were not interchangeable , both machines differentiated patients from controls with statistical significance . 
 structure - function analysis results were comparable , when either oct was compared to sap . 
1. Introduction
2. Materials and Methods
3. Statistical Analysis
4. Results
5. Discussion