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(Investigative Ophthalmology and Visual Science. 2000;41:2960-2966.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Ultrasonographic Evaluation of Optic Disc Swelling: Comparison with CSLO in Idiopathic Intracranial Hypertension

Ciro Tamburrelli, Tommaso Salgarello, Carmela Grazia Caputo, Andrea Giudiceandrea and Luigi Scullica

From the Institute of Ophthalmology, Catholic University, Rome, Italy.


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the accuracy and reproducibility of ultrasonographic (US) readings of optic disc elevations in patients with papilledema compared with confocal scanning laser ophthalmoscope (CSLO) measurements.

METHODS. One randomly selected eye of 22 patients with idiopathic intracranial hypertension (IIH) and a variable degree of optic disc swelling underwent five and three repeated measurements of disc height using high-resolution ultrasonography (Biovision unit; Quantel Medical, Clermont–Ferrand, France) and CSLO (Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering, Heidelberg, Germany), respectively. The same procedure was assessed in 14 subjects with variable degrees of physiologic optic disc cupping. US and HRT measurements from each group were individually compared with each other to estimate the accuracy of US readings in both disc conditions in comparison with HRT data.

RESULTS. Ultrasonographic readings were positively correlated with HRT measurements in both swollen (r = 0.62, P = 0.002) and excavated disc (r = 0.84, P < 0.0002). The 95% limits of agreement between the instruments were 0.24 ± 0.59 mm (mean ± 2 SD) and 0.05 ± 0.3 mm for swelling and cupping measurements, respectively. The coefficient of variation was 7.63% and 1.8% for swelling and 7.93% and 5.91% for cupping, with US and HRT, respectively.

CONCLUSIONS. The results indicate that US and CSLO readings are correlated in both disc swelling and cupping conditions, but to a different extent because of a significant discrepancy in papilledema. US assessment can be considered highly reproducible. Combined US and HRT optic disc analysis may be recommended in papilledema evaluation as long as a better correlation can be demonstrated in further studies.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An increased interest has grown around the possibilities of detection and assessment of glaucomatous cupping of the optic disc by means of ultrasonography. Some studies have attempted to test the ability of modern B-scanners to precisely detect disc cupping1 2 3 and a few parameters such as vertical and horizontal cup diameters.4 Results from these studies demonstrate a good agreement between ultrasonographic (US) measurements and ophthalmoscopic or laser scanning tomographic evaluation of disc cupping. On the other hand, US measurements of elevated optic nerve head have been suggested for use mainly in eyes with opaque media.5

Assessment of patients with swollen disc and idiopathic intracranial hypertension (IIH) includes morphologic evaluation of optic disc and retinal nerve fiber layer,6 7 8 9 10 11 12 visual acuity test,13 14 15 perimetry,7 9 10 15 electrophysiology,16 17 and radiologic studies such as computed tomography (CT) and magnetic resonance imaging (MRI) scans.18 19 Additionally, a defined role exists for US examination in these patients. Several papers stressed the importance of US transverse optic nerve diameter measurements to estimate the degree of intracranial hypertension in patients with papilledema,20 21 22 and a correlation was found between these readings and perimetric losses in patients with IIH.23 Recently, confocal scanning laser ophthalmoscope (CSLO) has been introduced to monitor papillary swelling in IIH.24 25 26 27

The present study was conducted to determine whether US readings of optic disc elevation, compared with CSLO measurements, can be considered accurate, reproducible, and useful in the detection and follow-up of papilledema.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Subjects
The patients enrolled in the study were recruited from larger cohorts of patients evaluated in both the Neuro-Ophthalmology and Glaucoma Services of our institution. Cases with refractive error less than -5.50 or more than +4.00 D spherical equivalent, astigmatism more than ±1.00 D, presence of disorders affecting the optic disc, or poor CSLO images (average variability >35 µm) were excluded.

Twenty-two patients (7 men, 15 women; mean age ± SD, 34.9 ± 14.7 years) with ophthalmoscopic evidence of variable degree of optic disc swelling and confirmed diagnosis of IIH were investigated. The diagnosis was based on the modified Dandy criteria for IIH.28 All the patients had a lumbar puncture documenting elevated cerebrospinal fluid (CSF) pressure (range, 260–310 mm H2O). Direct ophthalmoscopic and 90-D biomicroscopic assessment of the degree of papilledema was based on the staging scheme proposed by Frisén.8 The whole range of disc swelling (from stage 0: normal disc, to stage 5: marked disc swelling) was represented in the cohort of patients. Fourteen subjects (8 men, 6 women; mean age, 39.1 ± 17.8 years) with ophthalmoscopic evidence of variable degree of physiologic optic disc cupping were also enrolled in the study as a separate group.

One randomly selected eye of each subject underwent US measurements of the disc elevation or cupping with the calipers of the Biovision B-Scan "S" Vplus unit (Quantel Medical, Clermont–Ferrand, France). The same eye was then evaluated for disc morphometry by means of the Heidelberg Retina Tomograph (HRT; Heidelberg Engineering, Heidelberg, Germany). On two occasions within 3 days, five and three consecutive readings were taken for US and HRT evaluation, respectively. There was one operator only for US and one for HRT. Operators were masked to prior measurements and clinical information.

Informed consent was obtained from every subject or patient after the procedures used in the study were fully explained. The research followed the tenets of the Declaration of Helsinki.

Apparatus and Procedure
Ultrasonography.
We used a new generation B-scanner that has a focal length of 24 mm, a sector scan pattern probe, and a frequency emission of 10 MHz. At the beginning the disc area was examined with the probe placed in contact with temporal bulbar conjunctiva: a minimal angling of the probe provided a continuous display of vertical sections of the optic disc, dynamically scanned from the nasal to the temporal side. Similarly, horizontal sections of the disc were obtained by scanning from the superior to the inferior side, with the probe at the inferior bulbar conjunctiva. When the proper scanning plane was displayed, the image was frozen, and disc measurements were taken. A proper scanning plane is defined as the US section (horizontal or vertical) that displays the maximum disc elevation or excavation, excluding oblique sections of disc area that may turn in false large measurements. Its identification was accomplished with the probe placed as close as possible to the corneal limbus avoiding the lens and maximizing vitreous length. US measurements were performed where the echo profile of inner neuroretinal surface abruptly changed and delineated the convexity of the optic nerve head in case of elevation or the concavity in case of cupping. The measuring gates were placed on the frozen B-scan image.

In the elevated disc (Fig. 1) , the first gate was located on the leftmost edge echo signal of the swollen neuroretinal tissue protruding in the vitreous cavity and the second one on the leftmost edge of the strongly reflective echo line representing the lamina cribrosa, which is easily identified as a highly reflective and wide echogenic line located deeper in the disc.



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Figure 1. US transverse vertical scan of the swollen disc displaying the maximal elevation of the papilledema. Calipers are placed on top of the protruding swollen tissue (left) and on the strongly reflecting line corresponding to the lamina cribrosa (right).

 
In cupping, the first gate was placed on the point where the inner retinal surface changes direction toward the posterior concavity of the cup. Oblique measurements were avoided placing the second gate not on the lamina cribrosa, but at the same level in a point underlying the corresponding first gate on the inner retinal surface.

In all cases the distances were computed by setting the US unit at the standard velocity for soft tissue of 1550 m/sec, according to Ossoinig’s standardized guidelines.29 Such a velocity is commonly used for US assessment of the optic nerve. The earliest studies on the acoustic properties of normal human ocular tissues reported sound velocities ranging from 1506 to 1615 m/sec in brain and transversal or longitudinal optic nerve tissue examination (see Reference 30 for a review).

The mean of five readings was taken for each measurement. Test–retest variability of the five measurements, expressed by the average of the SDs of the biometric values in the five frozen images, was 0.09 ± 0.04 mm (range, 0.02–0.16 mm) for papilledema measurements and 0.06 ± 0.02 mm (range, 0.04–0.08 mm) for cupping measurements.

Confocal Scanning Laser Ophthalmoscopy.
The HRT was used to analyze the optic disc in a three-dimensional manner, in both disc swelling and cupping. HRT is a 670-nm, confocal scanning diode ophthalmoscope that acquires highly reproducible topographic images of the optic disc. A topographic image was taken as a series of 32 two-dimensional transverse optic section images, acquired approximately in 1.6 seconds, each consisting of 256 x 256 pixels (65,536 image elements). In this study three images were obtained for each eye, and the mean image of the three scans was used for the disc measurements, according to Weinreb et al.31 Details of this instrument and its reproducibility have been published either for papilledema or cupping analysis.31 32 33 34 35 36 37 38 39 The size of the field of view varied depending on the disc conditions evaluated: a 10° or 20° image was acquired for cupping or papilledema, respectively. The scanning depth of each topographic image series also varied, ranging from 0.5 to 4.0 mm in 0.5-mm increments, depending on individual disc morphology. Each mean topography image was automatically corrected for horizontal and vertical tilt.40 HRT software version 2.01 calculated the stereometric parameters of the optic nerve head. Magnification error was automatically corrected by using patients’ keratometry readings.40

For cupping evaluation, an optic disc contour line was manually drawn along the inner margin of the peripapillary scleral ring (Elschnig’s ring) on the HRT screen using a computer mouse system by a trained operator (TS), and the maximum cup depth (MxCD) was determined. This parameter is defined as the mean depth of the 5% of pixels with the highest depth values within the contour line, determined relative to the curved surface.40 This surface is bound by the disc contour line and follows the height variation of the retinal surface along the contour line, whereas the height of its center equals the mean height of the disc margin. Each section of the curved surface from its center to a boundary point is a straight line.40

For papilledema evaluation, a contour line was marked by the computer-generated circle outside the disc edema (Fig. 2) .25 26 37 This circle was located on healthy, not swollen retina, to avoid height measurement error, by having a reference surface as flat as possible (the HRT curved surface). For this purpose its mean radius was fixed to 2.5 mm for each eye—that is, along the extreme periphery of the image. The maximum height in contour (MxHC) parameter was determined. It is defined as the mean height of the 5% of pixels with the highest height values within the contour line, determined relative to the curved surface.40



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Figure 2. Swollen optic disc appearance by HRT topography and reflectance images (left and right, respectively). The contour line is shown along the periphery of the images.

 
Test–retest variability of the three measurements of each point, expressed by the average of the SDs of the topographic values of each pixel in the three images, was 16.43 ± 6.7 µm (range, 7.73–29.98 µm) for papilledema measurements and 19.58 ± 7.3 µm (range, 7.45–33.21 µm) for cupping measurements.

Statistical Methods
A linear regression analysis between the US and HRT observations was performed to investigate the strength of the relation between methods in both optic disc conditions. The accuracy of measurements was estimated from the variance of regression analysis parameters (i.e., slope, intercept, and correlation coefficient estimate).

The discrepancy between corresponding readings obtained by the two instruments was individually calculated, and the mean and SD were used to compute the 95% limits of agreement between the instruments (mean ± 2 SD).41 An independent Student’s t-test was used on the discrepancies in papilledema and cupping to assess the influence of different disc conditions on the agreement between US and HRT readings. To evaluate a possible influence of the size of the disc swelling or cupping on these discrepancies, a simple regression analysis between the difference of US and HRT readings and the subject mean was also performed.

The coefficient of variation was calculated as the square root of the mean value of the variance of the measurements obtained from each subject and then divided by the mean measured disc elevation or cup depth and reported in percent. We chose the coefficient of variation as a measure of reproducibility because it is straightforward and enables comparison of our results with those of other investigators.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The sets of demographic and clinical data (mean of repeated measurements ± SD) obtained from the study population with optic disc elevation (22 patients) and excavation (14 subjects) are reported in Tables 1 and 2 , respectively. In the first group (swelling) the height measurements ranged from 0.68 to 2.01 mm (1.17 ± 0.38 mm) and from 0.45 to 1.23 mm (0.93 ± 0.24 mm), with US and HRT, respectively. In the second group (cupping) the depth measurements ranged from 0.3 to 1.2 mm (0.79 ± 0.27 mm) and from 0.5 to 1.1 mm (0.74 ± 0.19 mm), with US and HRT, respectively.


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Table 1. Demographic and Clinical Data from the Optic Disc Elevation Group of Patients with IIH

 

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Table 2. Demographic and Clinical Data from the Optic Disc Excavation Group of Normal Subjects

 
Figure 3 depicts the individual tomographic MxHC values plotted as a function of the corresponding US readings in the disc-swelling group. The correlation was statistically significant (r = 0.62 ± 0.04 [SE], P = 0.002), indicating that MxHC tended to increase as US readings increased toward higher values. By linear regression analysis, the slope was 0.38 ± 0.02 (SE), and the y-intercept was 0.48 ± 0.03 (SE). A similar trend is displayed in Figure 4 , where the individual tomographic MxCD was plotted as a function of the corresponding US readings in the disc-cupping group. The correlation was statistically significant (r = 0.84 ± 0.03, P < 0.0002). By linear regression analysis, the slope was 0.57 ± 0.03, and the y-intercept was 0.29 ± 0.02. The line of equality in both plots shows that US readings tended to be larger than HRT measurements as the amount of heights or depths increased, especially in the disc-swelling group.



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Figure 3. HRT measurements of MxHC parameter compared with the corresponding US readings, individually obtained from the study population with optic disc elevation (n = 22; linear regression, r = 0.62, P = 0.002). The regression line and the identity line (dotted line) are also shown.

 


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Figure 4. HRT measurements of MxCD parameter compared with the corresponding US readings, individually obtained from the study population with disc excavation (n = 14; linear regression, r = 0.84, P < 0.0002). The regression line and the identity line (dotted line) are also shown.

 
The 95% limits of agreement between the two methods were larger in the disc-swelling group (0.24 ± 0.59 mm, mean ± 2 SD) than in the disc-cupping group (0.05 ± 0.3 mm). The discrepancies of US and HRT readings in the two optic disc conditions differed significantly (P < 0.05) by independent Student’s t-test. The correlation coefficients of the simple regression analysis (not shown) between the discrepancies and the subject mean were r = 0.54 (P < 0.01) in the disc-swelling group and r = 0.58 (P < 0.03) in the disc-cupping one, indicating that there was a statistically significant tendency for the mean difference to increase as the magnitude of the measurements increased.

Reproducibility expressed as coefficient of variation was 7.63% and 1.8% for swelling and 7.93% and 5.91% for cupping evaluation, with US and HRT, respectively.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IIH, also known as pseudotumor cerebri, is a disorder of elevated intracranial pressure of unknown cause. The diagnosis is mainly based on the exclusion of other pathologic conditions responsible for papilledema and/or headaches (according to modified Dandy criteria28 ). Thus, lumbar puncture documenting raised CSF pressure without abnormalities in protein level or cell count, and normal neuroimaging studies (CT and MRI)18 19 are necessary.

The possibility of visual loss is very real and demands a regular monitoring of patients with IIH for detecting functional15 and morphologic8 23 24 changes of the optic nerve. Despite some different evidences in the past,7 9 10 11 15 25 26 a recent report on patients with IIH with asymmetric papilledema has indicated that the amount of visual loss qualitatively correlates with the severity of disc edema, implying that visual loss in IIH is caused by papilledema and not by a retrolaminar mechanism.42 Therefore, either for diagnosis or follow-up, accurate examination of disc swelling may provide additional valuable information to the perimetric evaluation. In the modern clinical setting, accordingly to optic disc conditions, morphologic assessment is performed by common but very subjective methods such as direct ophthalmoscopy8 and stereoscopic slit lamp biomicroscopy using the 60-, 78-, or 90-D lens,43 44 or by the more detailed fluorescein angiography,6 retinal nerve fiber layer photographs,12 planimetry,45 46 stereophotogrammetry,47 and, mainly, recent computer-assisted technologies such as optical coherence tomography, scanning laser polarimetry, or confocal scanning laser tomography.39 The use of echography in the objective evaluation of the optic nerve head has been advocated by some investigators,1 2 3 4 5 and the accuracy and reproducibility of US readings have been assessed in relation to those of confocal scanning laser tomography by the HRT in disc cupping.4

We studied the reliability of US readings of disc elevation in patients with IIH evaluating the degree of agreement with measurements obtained with the HRT. The same evaluation was conducted in a group of subjects with ophthalmoscopic evidence of variable degree of physiologic disc excavation to verify our reliability by comparing these data with those from a recent report.4 The US measurements of disc elevation significantly correlated with the HRT readings, although a stronger correlation existed in disc cupping. The amounts of correlations, however, were not high for a comparison of measuring techniques, especially in the disc-swelling condition. Indeed, examination of the correlation coefficients (r = 0.62 and r = 0.84, for disc swelling and cupping, respectively) reveals that the two variables can only account for nearly 38% and 71%, respectively, of each other’s variance (r2). When evaluating the agreement instead of the strength of the relationship,41 a larger discrepancy between the methods was obtained for swelling (0.24 ± 0.59 mm, mean ± 2 SD) than for cupping evaluation (0.05 ± 0.3 mm), with a tendency for the US measurements to be higher than the HRT measurements with increasing magnitude of the examined structure. In contrast, the agreement decreased at both measurement extremes. The amount of the discrepancies in the two groups differed significantly (P < 0.05), suggesting that the optic disc conditions might contribute to the different degree of agreement. Specifically, in our opinion it may be explained, at least in part, by the reference planes used for measurements of cupping and papilledema in the two methods.

In cupping, the tomographic MxCD parameter measures the greatest vertical distance from the HRT curved surface (i.e., the peripapillary inner retinal surface) to the bottom of the excavation (the lamina cribrosa, approximately).40 The US method evaluates the distance between the profile of the inner retinal surface and the lamina cribrosa.2 4 Therefore, in cupping evaluation, both methods use similar planes.

In papilledema, the tomographic MxHC parameter measures from the maximum elevation of the disc to the curved surface (i.e., the retinal surface), because the lamina cribrosa is no more recognizable than it is in cupping. US readings are taken from the top of the elevated disc to the lamina cribrosa, which is still identifiable because of its higher reflectivity than the surrounding tissues. The distance between the inner retinal surface and the lamina cribrosa could account in part for the difference between the measurements obtained with US and HRT. The individual amount of such distance depends on the physiologic variability among optic disc excavations existing before the development of the edema.

However, there was much variation in the discrepancies among patients with IIH (Fig. 3) . In addition, the evidence of variation even among a few subjects of the cupping group (Fig. 4) suggests that the different agreement may be accounted for by factors other than the difference in the reference planes. Nevertheless, the 95% limits of agreement found in cupping depth evaluation were similar to those from a recent report4 (0.05 ± 0.3 versus 0.087 ± 0.328 mm), confirming the reliability of our procedure.

Analysis of individual data shown on the plots of Figures 3 and 4 with respect to the identity line indicates that there was a trend in the bias—that is, a tendency for the mean difference to increase as the magnitude of the measurements increased, in both conditions. Such behavior may depend on measuring errors owed to US or HRT accuracy or both.

The ideal perpendicular US scanning plane to the disc should pass through the lens, but it is not commonly used because refraction of the sound beam at the lens surfaces and nonpredictable angle of incidence on posterior pole occur. Therefore, the probe is placed as close as possible to the corneal limbus displaying the maximal vitreous length. In this case, the lens is avoided, but minimal obliquity may be introduced in the optic disc.

To the best of our knowledge, although sufficient accuracy of the HRT was reported on volumetric measurements of both excavations and elevations,35 only one study concerning the accuracy of a predecessor of the HRT on depth measurements has yet been published,48 and the accuracy on height measurements has never been investigated. Moreover, no study specifically evaluated accuracy of the HRT on both disc elevations and excavations of increasing magnitude. Variable accuracy may account for an additional measurement error especially affecting, in this study population, height measurements, which were larger than depth measurements. Additionally, even though we verified that the contour line was placed on flat retina avoiding the presence of humps on the contour line height variation diagram (i.e., small areas of local retinal edema under the contour line), mild retinal edema uniformly involving even the periphery of the tomographic image may be responsible for a slight underestimate of the MxHC parameter in large-sized papilledema.

Good reproducibility was found in cupping evaluation either for tomographic MxCD measurements (5.91%), which agrees with previous studies in which coefficient of variation ranged from 4% to 9%,32 34 49 50 or for US readings (7.93%). The similar US coefficient of variation found in swelling evaluation (7.63%) indicates that US assessment of swollen discs can be considered a highly reproducible method in observing patients with IIH and papilledema. Thus, besides the well-known role of echography in indirectly estimating CSF pressure by the determination of optic nerve sheaths diameters,21 22 the present study suggests a further potential usefulness of the technique in patients with IIH with ophthalmoscopic evidence of disc swelling. Serial contiguous B-scans (three horizontal and three vertical) of the optic disc from nasal to temporal and from superior to inferior margins should be recorded and used for measurements and follow-up. Obviously, longitudinal studies and improvements in both techniques, which may ameliorate correlation between the methods, are needed.

The differences between US and HRT readings in papilledema introduce a new parameter that may provide a good estimate of the full amount of disc edema: the true disc edema coefficient (TDEC). It represents the ratio between the ophthalmoscopically nonvisible edema, that is the space included between the planes of inner retina and lamina cribrosa (measurable as US-HRT), equivalent to the preexisting optic disc cup filled by the nerve fiber swelling, and the total height of the disc edema ultrasonographically measured: TDEC = (US-HRT)/US. Therefore, it indicates the contribution of the ophthalmoscopically nonapparent edema to the whole papilledema. TDECs higher than 0.5 suggest a greater amount of nonapparent than apparent edema, and vice versa.

To date, the relationship between functional damage and the amount of whole papilledema (as ultrasonographically measurable) has never been investigated. Moreover, because quantitative associations between functional and funduscopic assessment of papilledema in IIH have never been provided, there is no evidence of whether functional damage is related mostly to the ophthalmoscopically apparent or nonapparent edema. The potential ability of TDEC to separate different degrees of tissue edema, from disc cup disappearance to disc elevation, may be helpful in this purpose and, when associated with CSF pressure level assessment, relevant to the understanding of the pathophysiology of visual loss in IIH. Therefore, even the evaluation of TDEC changes during the resolution of papilledema may help analyze the relationships between papilledema and CSF pressure. Additionally, US evaluation of a possible anterior bowing of the lamina cribrosa related to direct transmission of elevated CSF pressure, currently attainable by new generation B-scanners, may provide further findings regarding this issue. Indeed, previous CT and MRI studies in patients with chronically high CSF pressure have postulated that the true edema has only a contributing role in disc elevation, indicating a bulging of the terminal optic sheath subarachnoid space into the posterior aspect of the globe at the optic nerve head as a major factor.51 52

Clinical implications for the TDEC may also be considered. For instance, patients with similar funduscopic disc elevation (Fig. 5) may differ in US measurements, and consequently in TDEC, suggesting a more serious condition and different clinical management for patients with higher values. Specifically, in spite of equal HRT heights, patients may show higher US values and TDEC, because of greater ophthalmoscopically nonapparent edema related to a more posterior lamina cribrosa position. Nonetheless, even the usefulness of TDEC needs a less variable discrepancy between these techniques.



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Figure 5. Representative drawing of two simulated cases with the same funduscopic disc elevation but different lamina cribrosa positions. The dashed, continuous, and dotted arrows represent the amount of US, HRT, and US-HRT measurements, respectively. Top left: The corresponding values of TDEC = (US-HRT)/US are also shown for cases A and B.

 
In conclusion, the present study, although indicating reproducibility of US readings and a correlation between US and HRT measurements, shows a significant discrepancy between the results of the methods of papilledema evaluation. The weakness of the agreement may be due in part to different reference planes. Further studies are needed to determine whether coupling HRT and US assessment of disc elevation may be clinically useful in the management of patients with IIH and papilledema. The use of US may be recommended in those patients in ophthalmologic centers where the HRT is not available and in presence of marked media opacities.


    Acknowledgements
 
The authors thank Benedetto Falsini for helpful suggestions in preparing the manuscript.


    Footnotes
 
Submitted for publication December 27, 1999; revised May 18, 2000; accepted May 24, 2000.

Commercial relationships policy: N.

Corresponding author: Ciro Tamburrelli, Istituto di Oftalmologia, Università Cattolica S. Cuore, Lgo F. Vito 1, 00168 Rome, Italy. citam{at}tiscalinet.it


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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