Normalized superior area: This parameter examines the data obtained exclusively in the superior part of the ellipse; higher value represents a physiological condition, while a lower value indicates an RNFL loss.

Normalized inferior area: This parameter examines the data obtained exclusively in the inferior part of the ellipse; higher value represents a physiological condition, while a lower value indicates an RNFL loss.

Ellipse standard deviation: It indicates the standard deviation of the values obtained in the calculation area.

Serial analysis

In the serial analysis are included up to four scansions of the same eye reported chronologically, in order to allow an evaluation of RNFL variations with time:

TSNIT confrontation graph: It compares, by means of a superimposition, the TSNIT graphs of two scansion of the same eye, taken during two consecutive visits. With this graph, it is possible to evaluate the variations of the RNFL thickness with time.

TSNIT serial analysis graph: It compares, by means of a superimposition, the TSNIT graphs of two, three, or four subsequent scansions of the same eye, taken during different visits. With this graph, it is possible to evaluate the variations of the RNFL thickness with time.

Deviation from reference map: It shows the variation of the RNFL thickness occurring during different visits. The colored areas and dots indicate the possible significant clinical variation. The colored legend defined variations with 20 mm increase.

Limits

The GDx, in the latest version called GDx-VCC, shows a high reproducibility with a good discrimination within normal and glaucomatous subjects (Greenfield et al., 2002; Weinreb et al., 2003;

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Brusini et al., 2005), and a close correlation between VF defects and RNFL damage (Bowd et al., 2003; Reus and Lemij, 2005).

However, the examination shows some limitation due to the ellipse localization that is operator dependent; furthermore, the interpretation of the results needs to be integrated with the clinical examination to achieve a precise diagnosis. There are still some limitations of the use of this instrument, for example, corneal refractive surgery (Choplin et al., 2005; Zangwill et al., 2005), lens surgery, presence of chorioretinal atrophy or scars, presence of myelinic fibers; all these conditions may interfere with birefringence. In addition, corneal opacity, pupil diameter lower than 2 mm, and significant vitreal opacities do not allow to perform the test.

The NFI can be considered positive when the value obtained is equal to or higher than 40 and this index has a sensitivity of 76.8% and a reproducibility of 89.1% (Colen et al., 2004).

Also, the progression of the damage should be considered with caution, since it was not demonstrated that variations from two consecutive examinations are certainly due to the progression of the disease rather than due to a physiological variation (Boehm et al., 2003).

The Heidelberg retinal tomograph

The HRT (Heidelberg Engineering, Heidelberg, Germany) is specifically designed to analyze the optic nerve head and gives an indirect evaluation of the RNFL. It is a confocal laser that uses a red diode laser of 670 nm wavelength. It performs a three-dimensional evaluation of the characteristics of the optic nerve head and peripapillary retina, with no need for mydriasis. It captures, in a time interval between 1.2 and 4.5 s, 32 optical pictures parallel to the retinal plane, analyzing the optic disk up to 151.

The scansions are obtained by a periodic deflection of the laser beam, by means of swinging mirrors, and using the confocal characteristics of the instrument, so that only the light coming from a determined focal plane is captured by the detector. The light coming from contiguous plans

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is discarded through two inner diaphragms, one of which is in front of the laser source and the other in front of the detector. In this way, the light from each retinal point is reflected toward the detector and is represented as a pixel on the screen. Each pixel height is automatically calculated with respect to the reference plane, located 50 mm behind the papillomacular bundle (Vihanninjoki et al., 2002).

A topographical image is acquired and, subsequently, a three-dimensional image of the optic nerve is obtained by computer analysis.

Before the image acquisition, it is important to upload patients’ personal and clinical data, corneal ray of curvature, and refraction: myopia and hyperopia of up to 11 diopters can be corrected, while high astigmatisms can be corrected by the use of adjunctive lenses. In patients with higher refractive errors, it is not possible to perform the examination.

At the start of the examination, the instrument makes an automatic scansion on 32 different planes on the papillary area (from a prepapillary to a retrolaminar plane) so that 32 bidimensional images are obtained.

The examination is repeated thrice so that three different images are obtained. From these images, the computer elaborates a mean topographic image 384 384 pixels wide, with three-dimensional reconstruction of the optic disk.

The instrument is able to evaluate the image quality by means of two quality parameters: interscan standard deviation (i.e., the mean test– retest variability: SD) and the mean confidence interval (CI) of the highest of the three images (good W20 mm, sufficient W50 mm). Furthermore, the instrument is able to give suggestions about the acquisition and to correct the scan depth and/or the refractive defects.

The mean image is shown with two maps: topographic map and reflectivity map. In the topographic map, the depth value is expressed with several colors (blue green is the deepest area; in the reflectivity map the reflectivity of each pixel is shown). On the topographic image, the operator can delimit the optic nerve head (contour line) along the inner border of the Elschnig’s scleral ring.

Fig. 8. HRT three-dimensional image of optic nerve head and peripapillary area.

Following this demarcation, the instrument automatically chooses a standard reference plane localized 50 mm under the mean peripapillary retinal thickness, along the contour line in the temporal sector between 3501 and 3561. This reference plane utilizes most part of the considered parameters circumscribed within the two areas of the optic disk: above the neuroretinal ring (green color) and below the excavation zone (red color).

The three-dimensional analysis allows obtaining planimetric and volumetric parameters (23 global and 13 partial) related to the optic nerve head and the RNFL measurement on the optical nerve head external border (Fig. 8).

The principal analyses obtained are the following:

Cross-section analysis: It allows to evaluate the three-dimensional aspect of the optic disk along one of the three Cartesian axes by means of a cursor.

Topographic map: It gives, in the absolute value or in mean7standard deviation, the height of each pixel analyzed by the system.

RNFL thickness diagram: It analyzes the thickness variation with double hump image from the temporal to the inferior sectors (TSNIT graph).

Stereometric parameters of the optic nerve head:

1.Dependent on the reference plane: cup area, cup/disk area ratio, rim area, cup volume, rim volume, RNFL crosssectional area, and mean RNFL thickness.

2.Independent of the reference plane: disk area, height variation contour, maximum

contour elevation and depression, CLM temporal superior and temporal inferior, mean cup depth, maximum cup depth, and cup shape measurement (morphological index of the cup or CSM) (Table 2).

Moorfields regression analysis (MRA): It compares the volumes of two stereometric parameters (rim and cup) in six papillary sectors with values obtained from normal

subjects and early glaucoma patients, with optic disk diameter between 1.2 and 2.8 mm2.

Data obtained from this classification give a good specificity and sensibility (Wollstein et al., 1998; Miglior et al., 2003) and can be shown in graphic and numerical details in comparison with the predictive values per age and optic disk diameter in the 95.0, 99.0, and 99.9% of the normative database. The graphs give quick visualization of the site and entity of the damage, evaluated according to a score in normal, borderline, and outline (Fig. 9).

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Interactive measurement: It gives an interactive horizontal and vertical profile of the optic nerve (Fig. 10).

Among the instrument functions, it is worth mentioning the possibility to study the glaucoma progression with time, which depends on the reproducibility of the several examinations performed.

The possibility to automatically display the previous contour line increases the reproducibility of the test (Verdonck et al., 2002; Tan et al., 2003) and, therefore, gives to the instrument the possibility to objectively analyze the papillary damage progression, by two different measurements:

1.Stereometric progression chart: Two sequential examinations are necessary; it also evaluates the variation of single stereometric parameters with time. The stability of the disease is indicated by the average normalized parameters value of 0, while the progression of the disease gives a value of 0.05. The

normalized parameters can be analyzed singularly and globally or with three different sector combinations: superotemporal sector (from 451 to 901), inferotemporal sector (from901 to 451), superior sector (from 22.51 to 112.51), inferior sector (from 112.51 to 22.51), superior hemisphere (from 01 to 1801), and inferior hemisphere (from 01 to1801). Studying the variation of single parameters, the instrument can evaluate a difference of values between baseline and follow-up (Fig. 11).

2.Progression analysis: Three sequential examinations are necessary. It is independent of the contour line and evaluates the modification of values from each pixel allowing the formation of the three-dimensional image (Chauhan et al., 2000). By studying the local variability, the test indicates if a change can be due to a modification of the parameter (change probability), which indicates a significant variation if lower than 0.05 compared with the basal examination.

In the progression analysis, the modified areas, with respect to previous evaluations, are shown with red pixels in the refractive map; a variation is considered significant if an area of at least 20 adjacent pixels is involved. Three examinations must be of excellent quality and perfectly aligned to perform the correct analyses, since the worst is the quality, the higher the variability.

It remains difficult to bring into evidence the damage progression because the criteria to state it are still not precise (Fig. 12). Recent studies have brought into evidence a long-term fluctuation of HRT parameters similar to that occurring with SAP (Chauhan et al., 2001; Funk and Mueller, 2003). The present version of the HRT, with the software 3.0 (HRT III), offers an option for alternative analysis that does not require placement of a contour line that also may introduce interoperator variability (Garway-Heath et al., 1999; Iester et al., 2001; Miglior et al., 2002).

Although the normative database in HRT II included 349 subject for the stereoscopic

parameters and 110 subjects for the MRA, the HRT III normative database included 733 healthy Caucasian eyes and 215 healthy African eyes (Burgansky-Eliash et al., 2007). Based on the enlarged database, the equations of the MRA were modified between HRT II and HRT III.

The technique provides stereometric data by applying an automatic model of the optic nerve

head shape, and the resultant morphological parameters are analyzed by a machine-learning classifier (relevance vector machine) resulting in a glaucoma probability score (GPS).

The GPS analysis provides a disease probability value based on the three-dimensional shape of the optic nerve and RNFL, and this classification represents the likelihood of glaucoma and not