data for this study were obtained from participants in the portland progression project , a prospective longitudinal study of the course and risk factors for glaucomatous progression . individuals with non - end - stage glaucoma or with ocular hypertension plus risk factors for glaucoma undergo testing with a variety of methods , including automated perimetry and sd - oct .
two separate cohorts were used : a  repeatability cohort ,  consisting of participants tested five times within a few months , and a  longitudinal cohort ,  consisting of participants tested approximately every 6 months over several years .
longitudinal data were used from the most recent eight visits at which reliable measurements ( as outlined below ) were acquired .
inclusion criteria for both cohorts were a diagnosis of primary open - angle glaucoma and/or likelihood of developing glaucomatous damage ( e.g. , high - risk ocular hypertension ) , as determined at the discretion of each participant 's physician .
exclusion criteria at entry included an inability to perform reliable visual field testing , best - corrected visual acuity worse than 20/40 , or other conditions or medications that may affect the visual field .
all protocols were approved and monitored by the legacy health institutional review board , and adhered to the health insurance portability and accountability act of 1996 and the tenets of the declaration of helsinki .
all participants provided written informed consent once all of the risks and benefits of participation were explained to them .
functional testing was performed using automated white - on - white perimetry with a humphrey field analyzer ( hfa ii ; carl - zeiss meditec , dublin , ca , usa ) , with a size iii stimulus , sita standard algorithm , and 24 - 2 test pattern .
visual field tests were excluded from analysis if they had greater than 33% false - negatives , 20% false - positives , or 33% fixation losses . for the primary analysis , mean deviation ( md )
was used , representing the global status of the visual field relative to age - appropriate normals , summarized across the 52 locations ( excluding the blind spot ) in the field . since it has been reported that the structure  function relation may be nonlinear , and that functional progression also may be nonlinear when expressed in decibels , a second summary functional measure was calculated .
the linear mean sensitivity ( lms ) was defined as the arithmetic mean of pointwise sensitivities expressed on a linear scale of 10 , so that 0 db  0.001 , 10
db  0.01 , 20 db  0.1 , and 30 db  1.0 . sensitivities on this scale then may be more linearly related to axon counts and , hence , to rnflt , and also may progress more linearly over time .
spectral - domain oct was performed with a spectralis oct ( heidelberg engineering , heidelberg , germany ) . on each test date , a circle scan was performed at a radius of 6 from the center of the optic disc as placed by the operator .
images were focused by the technician aiming to optimize the clarity of blood vessels within the rnfl .
follow - up scans were registered in real - time to the location of the baseline reference scan for each eye .
three segmentations were delineated on each circular b - scan , as shown in the example in figure 1 ; the inner limiting membrane ( ilm , in green ) , the posterior border of the rnfl ( in red ) , and the posterior border of the rpe / bruch 's membrane ( in yellow ) .
the instrument 's automated delineations were adjusted manually by experienced technicians when necessary to address obvious delineation errors ( without reference to the functional results ) .
the pixel corresponding to each of these three boundaries was recorded for each a - scan . in each case , this boundary pixel was assigned as belonging to the layer below the boundary ; hence pixels along the red line in figure 1 are counted not as part of the rnfl , but as part of the retinal ganglion cell layer .
three structures are delineated : the ilm ( green ) , posterior border of the rnfl ( red ) , and the posterior border of the rpe ( yellow ) .
since overall tissue reflectance varies between scans and between individuals , due to factors including differences in the exact focal plane and the quality of preretinal optics , normalization is required to better represent alterations of the tissues ' inherent optical properties rather than the influence of these imaging artefacts .
five methods for normalizing the reflectance intensity values were considered : ( 1 ) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels within the posterior vitreous ( i.e. , above the ilm ) , ( 2 ) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels coinciding with the delineated posterior border of the rpe ( the yellow line on fig .
1 ) , ( 3 ) the mean intensity of pixels within the rnfl divided by the mean intensity of a 5-pixel wide band immediately above the delineated posterior border of the rpe ( designed to be entirely within the high reflectance region of rpe visible on fig .
1 ) , ( 4 ) the mean intensity of pixels within the rnfl divided by the mean intensity of an 11-pixel wide band centered on the delineated posterior border of the rpe ( designed to be similar to the method used by dwelle et al . ) , and ( 5 ) the mean intensity of pixels within the rnfl divided by the mean intensity of sub - rnfl tissue , extending from one pixel below the posterior border of the rnfl to the posterior border of the rpe ( i.e. , between the red and yellow lines in fig .
1 ; chosen under the assumption that using a larger portion of the cross - sectional b - scan image should provide increased stability and , hence , lower variability , especially in the face of variability caused by the precise delineation of the retinal layers ) . using each of these normalizations in turn , an intensity ratio was calculated for each scan in the repeatability cohort .
the standard deviation of the five values for the scans obtained from each eye was calculated , and expressed as a percentage of the mean value .
this coefficient of variation can be considered equivalent to the reciprocal of the signal - to - noise ratio , under the assumption that the mean value is representative of the magnitude of the signal . to determine the most repeatable measure of reflectance intensity , these were compared between normalization techniques using the signed rank test for clustered data developed by datta and satten , since data from both eyes were used .
the per - eye rate of change over the most recent 8 visits in the longitudinal cohort was calculated by least - squares linear regression , for the two structural measures rnflt and reflectance intensity ratio , and for the two functional measures md and lms .
models then were formed to predict the rate of functional change ( by md or lms ) , using the concurrent rate of change of rnflt , the rate of change of the reflectance intensity ratio , and their interaction . since rates of change from both eyes were calculated , a generalized estimating equation ( gee ) model was used to account for correlation between the two eyes of an individual .
to assess whether disease severity affected the results , secondary analyses were performed after splitting the data into two halves , according to whether the final md in the visual field series for an eye was greater or less than the median value .
analyses were performed using the r language and environment for statistical computing ( version 2.15.3 ; r core team , vienna , 2013 , available in the public domain at http://www.r-project.org/ ) .
data for this study were obtained from participants in the portland progression project , a prospective longitudinal study of the course and risk factors for glaucomatous progression . individuals with non - end - stage glaucoma or with ocular hypertension plus risk factors for glaucoma undergo testing with a variety of methods , including automated perimetry and sd - oct .
two separate cohorts were used : a  repeatability cohort ,  consisting of participants tested five times within a few months , and a  longitudinal cohort ,  consisting of participants tested approximately every 6 months over several years .
longitudinal data were used from the most recent eight visits at which reliable measurements ( as outlined below ) were acquired .
inclusion criteria for both cohorts were a diagnosis of primary open - angle glaucoma and/or likelihood of developing glaucomatous damage ( e.g. , high - risk ocular hypertension ) , as determined at the discretion of each participant 's physician .
exclusion criteria at entry included an inability to perform reliable visual field testing , best - corrected visual acuity worse than 20/40 , or other conditions or medications that may affect the visual field .
all protocols were approved and monitored by the legacy health institutional review board , and adhered to the health insurance portability and accountability act of 1996 and the tenets of the declaration of helsinki .
all participants provided written informed consent once all of the risks and benefits of participation were explained to them .
functional testing was performed using automated white - on - white perimetry with a humphrey field analyzer ( hfa ii ; carl - zeiss meditec , dublin , ca , usa ) , with a size iii stimulus , sita standard algorithm , and 24 - 2 test pattern .
visual field tests were excluded from analysis if they had greater than 33% false - negatives , 20% false - positives , or 33% fixation losses . for the primary analysis , mean deviation ( md )
was used , representing the global status of the visual field relative to age - appropriate normals , summarized across the 52 locations ( excluding the blind spot ) in the field . since it has been reported that the structure  function relation may be nonlinear , and that functional progression also may be nonlinear when expressed in decibels , a second summary functional measure was calculated .
the linear mean sensitivity ( lms ) was defined as the arithmetic mean of pointwise sensitivities expressed on a linear scale of 10 , so that 0 db  0.001 , 10 db  0.01 , 20 db  0.1 , and 30 db  1.0 . sensitivities on this scale then may be more linearly related to axon counts and , hence , to rnflt , and also may progress more linearly over time .
spectral - domain oct was performed with a spectralis oct ( heidelberg engineering , heidelberg , germany ) . on each test date , a circle scan was performed at a radius of 6 from the center of the optic disc as placed by the operator .
images were focused by the technician aiming to optimize the clarity of blood vessels within the rnfl .
follow - up scans were registered in real - time to the location of the baseline reference scan for each eye .
three segmentations were delineated on each circular b - scan , as shown in the example in figure 1 ; the inner limiting membrane ( ilm , in green ) , the posterior border of the rnfl ( in red ) , and the posterior border of the rpe / bruch 's membrane ( in yellow ) .
the instrument 's automated delineations were adjusted manually by experienced technicians when necessary to address obvious delineation errors ( without reference to the functional results ) .
the pixel corresponding to each of these three boundaries was recorded for each a - scan . in each case , this boundary pixel was assigned as belonging to the layer below the boundary ; hence pixels along the red line in figure 1 are counted not as part of the rnfl , but as part of the retinal ganglion cell layer .
three structures are delineated : the ilm ( green ) , posterior border of the rnfl ( red ) , and the posterior border of the rpe ( yellow ) .
for each pixel within the circle scan , the intensity of the raw reflectance image was extracted using custom software .
since overall tissue reflectance varies between scans and between individuals , due to factors including differences in the exact focal plane and the quality of preretinal optics , normalization is required to better represent alterations of the tissues ' inherent optical properties rather than the influence of these imaging artefacts .
five methods for normalizing the reflectance intensity values were considered : ( 1 ) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels within the posterior vitreous ( i.e. , above the ilm ) , ( 2 ) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels coinciding with the delineated posterior border of the rpe ( the yellow line on fig .
1 ) , ( 3 ) the mean intensity of pixels within the rnfl divided by the mean intensity of a 5-pixel wide band immediately above the delineated posterior border of the rpe ( designed to be entirely within the high reflectance region of rpe visible on fig .
1 ) , ( 4 ) the mean intensity of pixels within the rnfl divided by the mean intensity of an 11-pixel wide band centered on the delineated posterior border of the rpe ( designed to be similar to the method used by dwelle et al . ) , and ( 5 ) the mean intensity of pixels within the rnfl divided by the mean intensity of sub - rnfl tissue , extending from one pixel below the posterior border of the rnfl to the posterior border of the rpe ( i.e. , between the red and yellow lines in fig .
1 ; chosen under the assumption that using a larger portion of the cross - sectional b - scan image should provide increased stability and , hence , lower variability , especially in the face of variability caused by the precise delineation of the retinal layers ) . using each of these normalizations in turn , an intensity ratio was calculated for each scan in the repeatability cohort .
the standard deviation of the five values for the scans obtained from each eye was calculated , and expressed as a percentage of the mean value .
this coefficient of variation can be considered equivalent to the reciprocal of the signal - to - noise ratio , under the assumption that the mean value is representative of the magnitude of the signal . to determine the most repeatable measure of reflectance intensity ,
these were compared between normalization techniques using the signed rank test for clustered data developed by datta and satten , since data from both eyes were used .
the per - eye rate of change over the most recent 8 visits in the longitudinal cohort was calculated by least - squares linear regression , for the two structural measures rnflt and reflectance intensity ratio , and for the two functional measures md and lms .
models then were formed to predict the rate of functional change ( by md or lms ) , using the concurrent rate of change of rnflt , the rate of change of the reflectance intensity ratio , and their interaction . since rates of change from both eyes were calculated , a generalized estimating equation ( gee ) model was used to account for correlation between the two eyes of an individual .
to assess whether disease severity affected the results , secondary analyses were performed after splitting the data into two halves , according to whether the final md in the visual field series for an eye was greater or less than the median value .
analyses were performed using the r language and environment for statistical computing ( version 2.15.3 ; r core team , vienna , 2013 , available in the public domain at http://www.r-project.org/ ) .
the repeatability cohort consisted of 53 eyes of 27 participants ( one eye was excluded due to an ocular pathology unrelated to glaucoma ) , tested 5 times per eye within a relatively short period of time . the longitudinal cohort ( restricted to eyes with at least 8 visits at which data of sufficient quality were obtained for sd - oct and perimetry ) consisted of 310 eyes of 205 participants .
the median rate of change of md ( from linear regression ) in the longitudinal cohort was 0.09 db / y , similar to a report from a clinical population of patients with glaucoma .
summary of the characteristics of the two cohorts table 2 summarizes the per - eye coefficients of variation within the repeatability cohort for each of the five normalization techniques considered . using the entire region from the posterior border of the rnfl down to the posterior border of the rpe ( i.e. , between the red and yellow lines on fig . 1 ,
method 5 in the list above ) had significantly lower coefficients of variation ( using the signed - rank test for clustered data ) than normalizations based on the posterior vitreous ( p = 0.011 ) or any of the three methods that relied upon rpe - based normalization ( all p < 0.001 ) .
therefore , method 5 was used to define the reflectance intensity ratio when analyzing data from the longitudinal cohort . the 95% confidence interval for test
the effect of the chosen normalization on intertest variability in the repeatability cohort is illustrated in figure 2 .
note that for comparison , rnflt had an average coefficient of variation in this cohort of just 1.3% ( range , 0.3%4.5% ) .
summary of the per - eye coefficients of variation ( i.e. , sd divided by mean ) of reflectance intensity ratio , using five different normalization methods the effect of normalization on reflectance intensity measures in the repeatability cohort . for each eye ,
the box extends from the second lowest to the second highest measurements ; the whiskers extend to the minimum and maximum measurements for that eye .
after normalization , the within - eye variability ( shown by width of each box ) is reduced as a proportion of the between - eye variability .
therefore , it can be seen that normalization based on the average reflectance of pixels between the nerve fiber layer and rpe greatly improves test  retest variability in this cohort , for which no true change should have occurred .
the mean of the chosen reflectance intensity ratio across all eyes and all visits in the longitudinal cohort was 2.69 ( sd , 0.99 ; range , 1.017.28 ) .
the mean per - eye rate of change of the reflectance intensity ratio in this cohort was 0.06 y ( sd , 0.22 y ; range , 0.90 to + 1.13 y ) .
the rate for a given eye was used together with the rate of change of rnflt to predict the rate of functional change . in univariate analyses ,
the rate of rnfl thinning was predictive of the rate of md change , with p < 0.0001 ; d / dt md = 0.062 + 0.096  d / dt rnflt ( where d / dt x represents the rate of change of measure x over time ) .
the value of d / dt md is measured in db / y , and d / dt rnflt is measured in m / y .
the rate of change of the reflectance intensity ratio was not by itself significantly predictive of the rate of md change ( p = 0.116 ) .
however , in a multivariable model , the interaction between the two structural rates of change improved upon predictions of the rate of functional change made using the rate of rnflt change alone : 

  in this model , d / dt rnflt had p = 0.0005 and the interaction term had p = 0.038 .
as an example of the effect size : the average value of d / dt rnflt from the longitudinal cohort was 0.75 m / y .
for this rate of rnfl thinning , if the reflectance intensity ratio changed at + 1.13 y ( the highest value observed ) , then the predicted rate of functional change would be d / dt md = 0.04 db / y . by contrast , if the reflectance intensity ratio changed at 0.90 y ( the lowest value observed ) then the predicted rate would be d / dt
md = 0.19 db / y , representing nearly a five times more rapid rate of functional deterioration . when sensitivities were transformed from db to a linear scale , results generally were similar .
d / dt rnflt was predictive of d / dt lms , with p = 0.0001 , but the rate of change of reflectance intensity ratio was not significantly predictive , with p = 0.256 . however , in the multivariable model , 
 where d / dt rnflt had p = 0.002 and the interaction term had p = 0.009 . 
 
secondary analyses were performed after splitting the cohort into two equal parts based on the final md in each eye 's series . among those eyes with final md
worse than 0.15 db ( the median value in the cohort ) , in the same multivariable model as before , d / dt rnflt still was a significant predictor of d / dt md with p = 0.0024 , but the interaction term had p = 0.650 .
by contrast , among eyes with final md greater than 0.15 db , d / dt rnflt had p = 0.1092 , while the interaction term had p = 0.0653 .
clearly these results are not definitive , since they did not reach statistical significance ( possibly due to the reduced sample size ) , but they hint that changes in reflectance intensity may be most useful at the earliest stages of the disease process .
the repeatability cohort consisted of 53 eyes of 27 participants ( one eye was excluded due to an ocular pathology unrelated to glaucoma ) , tested 5 times per eye within a relatively short period of time . the longitudinal cohort ( restricted to eyes with at least 8 visits at which data of sufficient quality were obtained for sd - oct and perimetry ) consisted of 310 eyes of 205 participants .
the median rate of change of md ( from linear regression ) in the longitudinal cohort was 0.09 db / y , similar to a report from a clinical population of patients with glaucoma .
table 2 summarizes the per - eye coefficients of variation within the repeatability cohort for each of the five normalization techniques considered . using the entire region from the posterior border of the rnfl down to the posterior border of the rpe ( i.e. , between the red and yellow lines on fig . 1 ,
method 5 in the list above ) had significantly lower coefficients of variation ( using the signed - rank test for clustered data ) than normalizations based on the posterior vitreous ( p = 0.011 ) or any of the three methods that relied upon rpe - based normalization ( all p < 0.001 ) .
therefore , method 5 was used to define the reflectance intensity ratio when analyzing data from the longitudinal cohort . the 95% confidence interval for test
the effect of the chosen normalization on intertest variability in the repeatability cohort is illustrated in figure 2 .
note that for comparison , rnflt had an average coefficient of variation in this cohort of just 1.3% ( range , 0.3%4.5% ) .
summary of the per - eye coefficients of variation ( i.e. , sd divided by mean ) of reflectance intensity ratio , using five different normalization methods the effect of normalization on reflectance intensity measures in the repeatability cohort . for each eye ,
the box extends from the second lowest to the second highest measurements ; the whiskers extend to the minimum and maximum measurements for that eye .
after normalization , the within - eye variability ( shown by width of each box ) is reduced as a proportion of the between - eye variability .
therefore , it can be seen that normalization based on the average reflectance of pixels between the nerve fiber layer and rpe greatly improves test  retest variability in this cohort , for which no true change should have occurred .
the mean of the chosen reflectance intensity ratio across all eyes and all visits in the longitudinal cohort was 2.69 ( sd , 0.99 ; range , 1.017.28 ) .
the mean per - eye rate of change of the reflectance intensity ratio in this cohort was 0.06 y ( sd , 0.22 y ; range , 0.90 to + 1.13 y ) .
the rate for a given eye was used together with the rate of change of rnflt to predict the rate of functional change . in univariate analyses ,
the rate of rnfl thinning was predictive of the rate of md change , with p < 0.0001 ; d / dt md = 0.062 + 0.096  d / dt rnflt ( where d / dt x represents the rate of change of measure x over time ) .
the value of d / dt md is measured in db / y , and d / dt rnflt is measured in m / y .
the rate of change of the reflectance intensity ratio was not by itself significantly predictive of the rate of md change ( p = 0.116 ) . however , in a multivariable model , the interaction between the two structural rates of change improved upon predictions of the rate of functional change made using the rate of rnflt change alone : 

  in this model , d / dt rnflt had p = 0.0005 and the interaction term had p = 0.038 .
as an example of the effect size : the average value of d / dt rnflt from the longitudinal cohort was 0.75 m / y .
for this rate of rnfl thinning , if the reflectance intensity ratio changed at + 1.13 y ( the highest value observed ) , then the predicted rate of functional change would be d / dt md = 0.04 db / y . by contrast , if the reflectance intensity ratio changed at 0.90 y ( the lowest value observed ) then the predicted rate would be d / dt md = 0.19 db / y , representing nearly a five times more rapid rate of functional deterioration . when sensitivities were transformed from db to a linear scale , results generally were similar .
d / dt rnflt was predictive of d / dt lms , with p = 0.0001 , but the rate of change of reflectance intensity ratio was not significantly predictive , with p = 0.256 .
however , in the multivariable model , 
 where d / dt rnflt had p = 0.002 and the interaction term had p = 0.009 . 
 
secondary analyses were performed after splitting the cohort into two equal parts based on the final md in each eye 's series . among those eyes with final md worse than 0.15 db ( the median value in the cohort ) , in the same multivariable model as before , d / dt rnflt still was a significant predictor of d / dt md with p = 0.0024 , but the interaction term had p = 0.650 .
by contrast , among eyes with final md greater than 0.15 db , d / dt rnflt had p = 0.1092 , while the interaction term had p = 0.0653 .
clearly these results are not definitive , since they did not reach statistical significance ( possibly due to the reduced sample size ) , but they hint that changes in reflectance intensity may be most useful at the earliest stages of the disease process .
retinal nerve fiber layer thickness remains an excellent structural measure for use in the diagnosis and monitoring of glaucoma .
it is highly repeatable ; the average per - eye coefficient of variation in our repeatability cohort was 1.3% , which is similar to the 1.9% intravisit coefficient of variation reported previously for the cirrus oct ( carl - zeiss meditec , inc . ) . in our longitudinal cohort ,
longitudinally , by contrast , the correlation between the rates of change of rnflt and md was only 0.361 . in part , this is because the range of values is narrower , and so the value of the correlation coefficient is reduced .
however , it also suggests that other sources of information are needed to refine predictions of longitudinal functional change .
as shown here , the reflectance of the rnfl appears to provide one such source .
it is possible that reflectance will prove to be even more useful in eyes with very early damage and/or ocular hypertension .
importantly , reflectance information can be extracted from existing sd - oct scans without hardware modifications .
it is important to note that the reflectance intensity ratio by itself was not predictive of functional change .
instead , it provides a method that may be able to refine predictions made using rnflt .
longitudinal changes observed in the reflectance must be considered in the context of changes observed in the rnflt , and not interpreted in isolation .
it certainly is possible that the measure of reflectance intensity used here might be improved upon .
most commercial oct instruments are optimized to visualize cross - sectional ( b - scan ) images and to measure the thickness of structures in the retina , not their reflectance .
for example , improvements could involve removing the effects of vessel shadows , and/or taking into account the directionality of retinal reflectance .
directionality is particularly important for cylindrical structures , such as axons and their aligned cytoskeletal components , which our normalization procedures , therefore , do not address .
indeed , the test  retest variability of our measure shown in figure 2 still is more than 10 times higher than the variability of rnflt measurement .
improvements in the repeatability of the reflectance intensity ratio would likely improve its use for prediction of functional change . in this study ,
the interaction between reflectance and thickness improved prediction of the rate of md change , but this was only just significant with p = 0.038 . accordingly , the standard deviation of residuals ( i.e. , predicted rate minus observed rate ) was reduced by less than 5% by the inclusion of reflectance intensity ratio .
if the test  retest variability of reflectance intensity could be improved further , then we would anticipate this p value being reduced .
moreover , commercial instruments may apply automatic gain controls to maintain signal strength in a more ideal range , providing benefits to imaging a broader range of eyes , but potentially confounding reflectance intensity measurements ( especially those without some internal normalization ) .
we also would note that it is possible that reflectance intensity may change before or after rnfl thinning .
this study did not incorporate any potential time lag between the observed changes in the retina , since each measure was assumed to change linearly over time .
furthermore , we looked solely at concurrent rates of functional change ; the aim was to predict the rate of functional loss during that same time period , rather than to predict subsequent functional loss .
previous studies have used a narrow band around the rpe to normalize rnfl reflectance , but used multiple b - scans at different radial distances from the center of the optic nerve head . by contrast , in this study a single circle scan at 6 from the center of the optic nerve head was used .
this is consistent with the most common current clinical approach , but means fewer pixels of information are available ( 1536 for this analysis ) and a more limited area of rnfl is assayed by the single circular scan . using a thicker band of axial information extending all the way from the posterior border of the rnfl down to the rpe
increased the number of available pixels , and so appears to give a more reliable measure of sub - rnfl reflectance when a single circle scan is used .
correspondingly , the test  retest variability of the reflectance intensity ratio was lower when using this thicker band
. it should be noted , though , that while we tested several alternative methods for interscan normalization , these do not represent an exhaustive examination of all possibilities , and further improvement may be possible .
the data used in this study consist primarily of cases of early , well - managed glaucoma .
only 4 of the 53 eyes in the repeatability cohort , and 20 of 310 eyes in the longitudinal cohort , had md worse than 6 db , a value often taken to indicate moderate functional damage .
approximately half of the eyes did not yet have significant functional damage when assessed by md alone .
therefore , this represents an extremely important clinical scenario , where early signs of functional progression are sought .
indeed , the secondary analyses hint that reflectance intensity may be more useful in  preperimetric glaucoma 
than in eyes with established functional defects , although we can not conclude this definitively at the present time . however , our conclusions have not been tested in cases of more severe glaucoma , and so it is not yet known whether reflectance intensity remains a useful prognostic measure later in the disease process or whether it represents a very early stage of structural damage .
we also would note that this study aims to predict the rate of change of md , because we sought a single continuous variable as the outcome measure to increase the statistical power of the analysis ; yet this is known not to be a particularly sensitive measure of visual field change , especially early in the disease process .
future studies are needed that examine pointwise progression , and/or with much larger cohorts that would enable assessment using binary definitions of progression and stability .
this study should be considered a  proof of principle ,  rather than providing a measurement that is ready to be implemented into commercially - available instruments , given the high test 
however , the principle behind the measurement is something that can be qualitatively assessed already , even if quantitative assessment is not yet clinically available .
figure 3 shows a magnified portion ( for easier visualization ) of the peripapillary rnfl b - scan shown in figure 1 ; together with the corresponding portion of a scan from the same eye 18 months later . on the date of the first ( upper ) scan ,
this participant had an average rnflt of 91.2 m and a reflectance intensity ratio averaged around the entire circle scan of 4.73 . using the variability estimate from table 2 , this means that the 95% confidence interval for test 
retest is ( 3.17 , 6.29 ) . by the date of the second ( lower ) scan ,
the nerve fiber layer is visibly less reflective in the lower panel . within the same time period ,
the md decreased from 5.0 db on the date of the first scan to 7.6 db on the date of the second scan .
the results of this study suggested that clinicians should be on the lookout for similar decreases in rnfl reflectance , as they may correspond ( as in this example ) to worsening function .
example of a section of an oct scan ( top ) , and the same section 18 months later ( bottom ) . in that time period ,
this corresponded to worsening function during that same period . in practice , light intensity and
focus may vary between scans , with the result that changes in reflectance will not always be easily visible to the clinician .
furthermore , to map the raw intensity information to a range compatible with most common monitors and/or clinical printouts , raw intensity values are compressed to 256 grayscale levels ( 8-bit range ) , reducing the precision available . our normalization process ( which is applied to raw intensity data ) may not be easy to implement qualitatively while reviewing a series of scans in a clinical setting .
therefore , it is hoped that instrument manufacturers could eventually add such measures to their output , enabling more sensitive detection of changes in reflectance . in summary , we described a method to quantify the reflectance intensity of the rnfl from sd - oct scans . for a given rate of rnfl thinning , a reduction in rnfl reflectance is associated with more rapidly deteriorating function .
further work will examine whether localized changes in rnfl reflectance correspond to localized functional deterioration .
the causes of these reductions in reflectance intensity are not entirely clear , but they raise the intriguing possibility of dysfunctional yet surviving ganglion cell axons , which may be candidates for neuroprotection or rescue .
clinically , we would recommend examination of the images produced by oct instruments , as they may reveal important changes that are not apparent from looking at rnfl thickness or other structural measures in isolation .
 purposewe determined whether longitudinal changes in retinal nerve fiber layer ( rnfl ) reflectance provide useful prognostic information about longitudinal changes in function in glaucoma.methodsthe reflectance intensity of each pixel within spectral - domain optical coherence tomography ( sd - oct ) circle scans was extracted by custom software . a repeatability cohort comprising 53 eyes of 27 participants ( average visual field mean deviation [ md ] 1.65 db ) 
 was tested five times within a few weeks . to minimize test  
 retest variability in their data , a reflectance intensity ratio was defined as the mean reflectance intensity of pixels within the rnfl divided by the mean between the rnfl and rpe . 
 this was measured in a separate longitudinal cohort comprising 310 eyes of 205 participants tested eight times at 6-month intervals ( average md , 0.99 db ; median rate of change , 0.09 db / y ) . 
 the rate of change of this ratio , together with the rate of rnfl thinning , and their interaction , were used to predict the rate of change of md.resultsin univariate analyses , the rate of rnfl thinning was predictive of the rate of md change ( p < 0.0001 ) , but the rate of change of reflectance intensity ratio was not ( p = 0.116 ) . however , in a multivariable model , the interaction between these two rates significantly improved upon predictions of the rate of functional change made using rnfl thickness alone ( p = 0.038).conclusionsfor a given rate of rnfl thinning , a reduction in the rnfl reflectance intensity ratio is associated with more rapid functional deterioration . incorporating sd - oct reflectance information may improve the structure  function relation in glaucoma . 
Methods
Population
Testing Protocol
Measurement of Reflectance Intensity
Prediction of Functional Change
Results
Population
Measurement of Reflectance Intensity
Prediction of Functional Change
Discussion