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Year : 1994  |  Volume : 42  |  Issue : 2  |  Page : 89-99

Diagnostic tests for corneal diseases

Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Mahipal S Sachdev
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029
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Source of Support: None, Conflict of Interest: None

PMID: 7927639

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Sachdev MS, Honavar SG, Thakar M. Diagnostic tests for corneal diseases. Indian J Ophthalmol 1994;42:89-99

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Sachdev MS, Honavar SG, Thakar M. Diagnostic tests for corneal diseases. Indian J Ophthalmol [serial online] 1994 [cited 2019 Aug 20];42:89-99. Available from:

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Over the past several years there has been increased research and growing interest in the field of corneal diseases. Our approach to the diagnosis of corneal disorders has undergone a total transformation since the introduction of specific tests to detect structural and functional abnormalities of the cornea. These give surgeons more critical and accurate information for early and better diagnosis and help in decisions concerning their management. In a clinical setting, corneal diseases can manifest as an alteration in topography, transparency change in the endothelial cell morphology and function, loss of epithelial integrity, loss of sensation, opacification, and vascularization. Early diagnosis and institution of specific therapy, and quantification for gauging clinical response are mandatory for the optimal management of corneal diseases. We have attempted to describe the currently available investigative modalities to diagnose and quantify corneal responses to various diseases. The choice of the method of diagnosis, however, depends on a variety of factors, including availability, reliability, reproducibility, cost, and the other infrastructure facilities. Diagnostic tests available for the structural and functional evaluation of corneal diseases are classified as shown in [Table - 1].


Slit-lamp Biomicroscopy

Of all the ocular structures, the cornea is the most accessible to complete clinical examination due to its anatomic location and transparency. The basic and the most useful instrument available for refined examination of all the layers of the cornea is the slit­lamp biomicroscope. Berliner has described the following six basic methods of slit-lamp examination:

Diffuse illumination to assess general topography and gross alterations

Sclerotic scatter for subtle epithelial or stromal irregularities

Direct focal illumination for the accurate determination of the depth of lesions

Retroillumination to evaluate fine epithelial alterations, stromal opacities and keratic precipitates

Indirect illumination to detect lesions like vascularization

Zone of specular reflection for the gross assessment of endothelial cell pattern

In addition to standard biomicroscopy illumination techniques, the use of cobalt blue illumination helps to detect a Kayser- Fleischer ring, while red-free illumination highlights corneal nerves and subtle alterations in the Descemet's layer. Slit-lamp biomicroscope also provides a base for the use of accessories like optical chymeter.


1. Tests to Detect Structural Abnormalities

a. Corneal Topography

Cornea, which is the major refracting surface of the eye, provides +43.0 diopters of convergent lens power. Therefore, relatively small distortions in the topography of the cornea can degrade visual acuity. Evaluation of corneal topography is becoming an integral part of the corneal surgeon's practice of ophthalmology, where it is being used to guide selective suture removal following keratoplasty and cataract surgery; to make an early diagnosis of corneal shape anomalies such as keratoconus, pellucid marginal degeneration, and Terrien's marginal degeneration; to evaluate and guide the results of refractive surgical techniques; to determine simple or irregular astigmatic refraction; and to aid in the fitting of contact lenses. Corneal topography can be assessed by keratometry, keratoscopy, and videokeratography.

(i) Keratometry: The standard instrument for the measurement of corneal surface irregularities is the keratometer, which measures the corneal curvature based on dioptric values obtained at four points across two perpendicular chords, approximately 3 mm apart, on a mire which is reflected off the anterior tear film of the cornea. One pair of points is aligned along the steepest axis of the corneal surface, with the second pair along the axis perpendicular to the steepest axis. These two readings yield radius of curvature values which are used to approximate the refractive power (K values) of the apical zone of the cornea. The keratometer has been designed to take into account the combination of anterior plus power and the posterior minus power of the cornea. It is important for the clinician to realize that what is being measured is not the true refractive power of the anterior surface but refractive power of the entire cornea.

Of three prototype models of manual keratometer available, the Bausch & Lomb keratometer is the most popular one, mainly due to its simplicity. The instrument is adjusted to bring the mires into focus, and is rotated until the mires are aligned to determine the axis of the cylinder. The plus signs are then superimposed to determine the radius in the horizontal meridian, and the minus signs are superimposed to determine the curvature in the vertical meridian. The refractive power of the cornea in the horizontal and the vertical meridian with the axis of each meridian is directly read out when the end point is reached. The American Optical and Haag-Streit instruments are used in a similar fashion. The measuring range of a keratometer extends from +36 to +52 diopters. In extreme cases, the range can be extended by addition of low power lenses in front of the objective; from +30 to +61 diopters. The computerized autokeratometer is much simpler to use, is more accurate and gives highly reproducible results. A hand-held autokeratometer [Figure - 1] provides the convenience of portability along with the inherent ease and accuracy of autokeratometer.

Although for many years the keratometer has been the principal measuring device used for determining central corneal curvature, and has been providing highly repeatable results, it has certain shortcomings:

• The keratometer is incapable of measuring the corneal curvature inside or outside of the 3 mm apical zone, as a result evaluation of the midperipheral and peripheral zones is not possible.

• The assumption that the surface of the cornea is an orthogonally symmetric spherocylinder, based on which the K values are reported, is not consistent with the known asymmetries of the cornea and actual aspheric shape. Modifications of the original keratometer to measure the peripheral corneal curvature by incorporating peripheral-fixation devices (topogometer) and small-mire keratometry have failed to meet the demands of the day.

(ii) Keratoscopy and Photokeratoscopy: The first attempt at determining the shape of the corneal surface was based on a concept developed in 1880 by Placido who introduced the use of a disk with equally placed concentric white rings on a black background, now known as the 'Placido disk'. The reflected image of the disk is observed through a hole in the centre of the innermost ring. Deviations of the corneal shape appear as distortions in shape or concentricity of the rings. This method provides the observer with qualitative information about the patient's corneal curvature.

Photokeratoscopes are devices for photographing the Placido ring image reflected from the corneal surface [Figure - 2]. Photokeratoscopy offers a distinct advantage over keratometry in that data are accumulated from a large area (9 to 12 mm) of the corneal surface. The steps in the qualitative analysis of keratoscopic photographs are include examination for artifacts, examination of central mires to estimate the central corneal power and astigmatism, and examination of the peripheral mires.

The keratoscope, however, is a qualitative device and subtle, but visually significant alterations are often not discerned. Attempts at computerized quantification of data points on the photokeratoscopic images and a three-dimensional reconstruction have proven to be slow, tedious and highly subjective to approximations and errors.

(iii) Computer-Assisted Videokeratography (CAVK): Computerized corneal topography [Figure - 3] is a logical advance from the basic principles of keratometry and photokeratoscopy. CAVK provides qualitative and quantitative data about the entire corneal surface; is quick, accurate, and reproducible; and provides a complete colour-coded representation of the entire corneal contour. Each colour in these maps represents a selected range of powers. Colours close to the blue spectrum are used to identify low powers and colours close to the red spectrum identify high powers. This type of presentation allows the observer to see complex patterns of power distribution, not always readily recognized by simple visual inspection of keratoscope photographs. Colour­coded representations of corneal contour could be in the form of a topographical map, which is currently the most preferred method of display; or in the form of an absolute chart, a comparative chart, an isometric profile, a normalized scheme, or a three-dimensional representation [Figure - 4]. Recent models of CAVK can also provide data in the form of absolute statistical indices such as surface regulatory index; surface asymmetry index; irregular astigmatism index; differential sector index; and keratoconus prediction index. One major area of utility for this type of a system is in the analysis of complex patterns of power distribution created by corneal ectatic disorders and in the planning, execution, and evaluation of keratorefractive procedures.

A number of other optical techniques such as Moir6 keratometry, interferometry, ultrasonography, stereo photogrammetry, profile photography, and holography have been evaluated to measure corneal topography. However, none of these approaches has proved thus far to Le sufficiently accurate or practical for clinical application.

b. Corneal Thickness

Pachymetry is the method of measuring corneal thickness. The normal corneal thickness increases irregularly towards the limbus where it ranges from 0.7 to 0.9 mm. The central corneal thickness is between 0.49 mm and 0.56 mm and readings of 0.7 mm or more are indicative of endothelial decompensation. Measurement of corneal thickness can be of value in:

• Evaluating the degree of corneal oedema in various states of corneal decompensation.

• Assessing the thinness of the cornea as in keratoconus.

• Following up patients with keratoplasty to determine endothelial cell function and its temporal recovery and to become alerted to graft decompensation.

Assessing the level of a corneal opacity.

• Surgical planning of keratorefractive procedures like radial keratotomy and astigmatic keratotomy.

The types of pachymeters in use are: Haag-Streit optical pachymeter; Ultrasonic pachymeter; Specular microscopic pachymeter; Laser pachymeter; and CAVK devices.

(i) Haag-Streit Pachymetry: Haag-Streit pachymeter type I, which is a standard slit-lamp attachment is the prototype of a corneal optical pachymeter. It comes with or without a Mishima-Hedbys fixation attachment, which ensures the perpendicularity of the incident beam on the corneal surface. The instrument contains two piano glass plates that split the image of the corneal parallelo-piped. A slit beam is projected perpendicularly to the cornea through the narrow diaphragm of the instrument. The observer, looking through a uniocular right-sided split-image eyepiece that replaces the regular eyepiece of the slit-lamp, moves the scale of the instrument until the focused upper half of the corneal image is positioned so that its posterior surface is in direct contact with the anterior surface of the lower image. The corneal thickness is then directly read from the scale on the instrument. The measurable range of an optical pachymeter is from 0 to 1.2 mm, with a least gradation of 0.02 mm. The problem with an optical pachymeter is its lack of accuracy in measurements; the usual range of error with an optical pachymeter is ± 2%.

(ii) Ultrasonic Pachymetry: In 1980, Kremer introduced the ultrasonic pachymeter, which made measurement of the corneal thickness easier because the probe tip could be placed perpendicularly on the cornea at any spot. Ultrasonic pachymetry was also more precise because it eliminates interobserver variations. It is easier for paramedical staff to use, portable, and can be used intraoperatively. The procedure is based on the measurement of the time difference between echoes of signal pulses reflected from the front and back surface of the cornea, the sound velocity through normal cornea being taken as 1640 m / sec. The tip of the probe must be applied within 10 degrees or less of perpendicularity in order to register return of the sound wave. The readings thus obtained are accurate from 5 to 10 µ.

(iii) Specular Microscopic Pachymetry, CAVK Pachymetry, and Laser Pachymetry: Specular microscopic pachymetry and CAVK pachymetry are not practical for routine use. They can, however, be used for pachymetry if specular microscopy or corneal topographic analysis are being performed in concurrence. Laser pachymetric devices are still being evaluated for possible clinical use.

c. Evaluation of Endothelial Cells

The human corneal endothelial cell layer consists of 350,000 to 500,000 compact cells per cornea at birth arranged in a monolayer 4 to 6 g thick. The cells have a uniform density, are compact, hexagonal, with the angle of intersection of cell sides being 120°, and cell borders being single-edged and straight. Older corneas may have a more varied cell morphology (pleomorphism) and variations in cell size (polymega­thism).

(i) Slit-lamp Aids: Assessment of the corneal thickness provides an indirect evidence of endothelial cell function. Slit-lamp biomicroscopic examination of the zone of specular reflection under high magnification yields some information about the endothelial status in terms of guttata and cell dropouts. Recently certain contact and non-contact lenses have been introduced (McIntyre's Grid; Eisiter's Lens), which can be used with slit-lamp biomicroscopes for a magnified, less- distorted view of the endothelial cells. Estimation of endothelial cell density is also possible with these devices.

(ii) Specular Microscope: The introduction of specular microscopy has made possible the direct comprehensive assessment of endothelial cell morphology in vivo. Undoubtedly the largest application for specular microscopy is the preoperative evaluation of the endothelial cell layer. However, there are other significant applications of specular microscopy viz. evaluation of endothelium as a diagnostic aid in disease states like Fuchs' endothelial dystrophy, iridocorneal endothelial syndrome, lymphoma, and uveitis; evaluation of donor corneas; and documentation of effects of intracameral drugs and contact lens use on the endothelium.

The first instrument was designed by Maurice in 1968, based on the principle of specular reflection of a small amount of light (0.02% of transmitted light) from the aqueous humour-endothelial cell interface. Any irregularities in the smooth surface such as intercellular borders, guttata, and swollen endothelial cells appear black.

The original specular microscope design utilized slit illumination, resulting in a gradient of contrast from the bottom to the top of the frame, which drastically limits the width of the image that is available for analysis.

Koester extended the basic principle of Maurice's scanning slit optical microscope to develop a scanning oscillating mirror which provides a wider field of view of over 1 sq mm of endothelial cells. The Keeler-Konan Poklington wide-field microscope was designed to neutralize the strong superficial corneal reflection by making the optical system from materials whose refractive index gradually decreases from the objective lens to the corneal surface. A video camera can also be attached for greater versatility of the instrument.

Both the standard and the wide-field specular microscopes are contact devices, involving the use of a contact cone in approximation to the corneal surface. A noncontact epithelial- endothelial specular micro­scope has been recently introduced which has the capability of grid method of cell count as well as complete quantitative analysis with the aid of built­in analytical software.

Interpretation of Endothelial Specular Microscopy

Qualitative Analysis

Cell size: Variations in cell size (polymegathism) could be age-related or could be an indirect manifestation of endothelial cell loss.

Cell borders: Normally appear as dark lines which are thin and straight. Endothelial cell dysfunction may lead to curved cell borders. Swelling of the endothelial cells can give rise to slight differences in the height of cell borders and simulate 'doubling' of the border. At a common intersection three adjacent cells usually meet at an angle of about 120°. An endothelial cell is normally surrounded by six other cells. Deviations and variations represent ageing changes or reparative processes after severe cell injury. The formation of clusters of radially arranged cells called rosettes are also indicative of the same.

Cell shape: In normal endothelium the hexagonal shape prevails. After a severe endothelial injury, a pleomorphic picture with bizarre cell-forms can result.

Cytoplasmic changes: Dark and light variegated cellular appearance represents intracellular vacuole formation.

Changes due to nonendothelial structures: Nonendothelial structures like hyaline material in pseudoexfoliation, leukocytes, and extracellular pigments outshine the endothelial reflex and appear bright. Suppression of the reflex due to interruption of the zone of specular reflection is caused by excrescences of the Descemet's membrane (Hassal­Henle warts) and inflammatory cells adherent to the endothelium. Dark, oval to round areas (guttata) result. Specular microscopic differentiation between the Descemet's warts and the inflammatory cells is difficult.

Quantitative Evaluation

The morphometric evaluation is done in two ways:

Fixed frame analysis: Endothelial cell count in a given area is usually expressed as density per 1 sq mm.

Estimation of cell size: For single cell size estimation, a usual planimeter or a semiautomatic image analyzer can be employed. Estimation of individual cell size allows morphometry in areas with only a limited number of visible cells.

A semiautomatic image analyzer Leitz-ASM allows determination of the single cell areas in square micrometers; their mean value with standard deviation and variation coefficients; the cell number in a given area; and frequency distribution of endothelial cell size [Figure - 5]. Automated morphometric analysis works best for high-contrast wide-field specular images. The latest morphometric technique is based on an analysis of the two- dimensional Fourier transform of the cell­boundary pattern, which can provide reliable measures of average cell size, cell size variation, and angular orientation characteristics of the cell patterns.

Epithelial Cell and Stromal Morphology

The specular microscope can be used for the morphological evaluation of the corneal epithelium [Figure - 6] and stroma. Epithelial cells can be evaluated under a variety of conditions and with vital stains. The epithelial cells take on a hexagonal appearance of light and dark areas. The healing epithelium shows a whorl- like pattern which is thought to be a natural physical pattern of cell replication from the periphery to cover the central area. Certain persistent ulcers reveal abnormal looking epithelium on specular microscopy and bullous keratopathy reveals blisters beneath the epithelial layer. The specular microscope can also be used to study the stroma. Corneal stromal deposits in granular, macular and lattice dystrophy have been visualized. Intracorneal foreign bodies and sutures, and the tissue distortion that they can create can be easily noted. The specular microscope can also evaluate the continuing physiological processes that occur in the cornea in response to the environment imposed on it by the surgeon, be it a graft, or a cornea covered by a contact lens [Figure - 7], or a cornea lying dangerously close to an intraocular lens.

(iii) Tandem Scanning Confocal Microscope (TSCM): The application of conventional optical imaging to study the morphology and function of the layers of the cornea in vivo is currently limited by resolution, depth of focus, and contrast. Current developments in optical microscopy involving confocal imaging are dramatically increasing resolution, contrast, and depth of focus by optically sectioning through structures without the need of specular surface reflections. The essential principle of confocal imaging is point illumination and detection, which is achieved by both the illuminating and objective lenses having the same focus. Confocal microscopes of two types have been developed - Laser Scanning Confocal Microscope (LSCM) and Tandem Scanning Confocal Microscope (TSCM). At the currently available scan rates for LSCMs, images are not generated in real­time, while TSCMs which have a modified Nipkow disk [Figure - 8] provide faster scan rates.

The TSCM developed for ophthalmic use provides high resolution and contrast images of living ocular tissue. Currently, the technique is most successfully used for detection of corneal structural abnormalities. TSCM images of the epithelium provide details about the number and shape of superficial epithelial cells and serial images of deeper layers, including the cell nuclei and the wing cells. Epithelial basal lamina - Bowman's membrane can also be visualized. Information about the cellular structure of the corneal stroma, inflammatory cells, and corneal nerves is provided by the TSCM. High resolution wide-field views of the corneal endothelium can also be seen even when the cornea is oedematous or the overlying stroma is irregular and opaque.

In addition to studying normal corneal structure and function in vivo, the TSCM also allows for the dynamic, sequential, noninvasive investigation of the wound healing process [Figure - 9] at the cellular level. Images generated by the TSCM can be captured and stored in real-time to provide quantitative assessment of the corneal structure.

d. Epithelial Integrity

Corneal epithelium, due to its direct and constant exposure to the environment is prone to develop abrasions. Infective and noninfective keratitis are also common. The integrity of the corneal epithelium in such a situation can be assessed and monitored by vital staining: Fluorescein staining and Rose bengal staining [Figure - 10].

(i) Fluorescein Staining: Fluorescein staining of the cornea involves the instillation of 2% sodium­ fluorescein solution either in the form of drops or in the form of a strip into the conjunctival sac. With blinking the fluorescein is well distributed in the cul­de-sac. Any area with epithelial defect takes up fluorescein, which, under cobalt blue illumination of the slit-lamp biomicroscope is well highlighted.

(ii) Rose Bengal Staining: A more sensitive staining method involves the use of rose bengal 1%o solution. Unlike fluorescein, rose bengal dye stains damaged epithelial cells even in the absence of discontinuity, and stains corneal filaments. Rose bengal staining secondary to dry eye states shows a typical pathognomonic pattern. It has only one disadvantage, i.e., irritation upon instillation.

e. Corneal Vessels

(i) Anterior Segment Fluorescein Angiography:

Although corneal vascularization is readily accessible to slit-lamp evaluation, anterior segment fluorescein angiography (ASFA) has been found to be useful. ASFA has also been found useful in some cases of congenital anomalies of the cornea. In posterior embryotoxon, the ciliary veins have been noted to appear abnormally small and a possible ischaemic component of this anomaly was postulated. Leakage of corneal vessels is a function of the degree of compactness of the surrounding tissues, and may serve as an indicator of the amount of activity of the inciting corneal disease. This is especially true in cases of chronic or periodically recrudescing inflammatory diseases such as stromal herpes simplex keratitis, trachoma, and interstitial keratitis.

(ii) Tandem Scanning Confocal Microscope: TSCM is also a very useful tool for the in vivo assessment of corneal vascularization.

2. Tests to Detect Functional Abnormalities

a. Endothelial Cell Function

(i) Spectral Fluorophotometry: Spectral fluorophotometry measures the permeability of the endothelium to fluorescein, thus reflecting the state of barrier function of the cells. Although it is not used routinely in clinical practice, fluorophotometry is a promising research tool for detailed analytical studies of endothelial function.

(ii) Redox Fluorophotometry: Redox fluorophotometry is a recent development. It can be used to assess cellular respiratory functions and therefore, cell viability' in diagnostic evaluations (post­surgical endothelial dysfunction; Fuchs' dystrophy) and in the selection of transplant donor material.

b. Corneal Sensivity

Corneal aesthesiometry is of frequent importance in examining cases involving lesions of the fifth nerve, cases with ulcerative keratitis and degenerative lesions. This can be done subjectively by touching the cornea lightly with the finely drawn-out end of a wisp of damp cotton-wool. The presence of normal sensations elicits a prompt blink reflex, but care should be taken to approach the cornea from the lateral side of the patient to prevent optical blinking response. A certain degree of quantitative discrimination can be imparted into this test by using the fellow cornea as control. In an attempt to quantify corneal sensations, vonFrey used animal hairs of varying degrees of stiffness with spring balances to calibrate the amount of force delivered. Cochet­Bonnet aesthesiometer is one of the commonly used devices for the quantification of corneal sensitivity. If the filament is applied perpendicularly to the corneal surface, most patients will feel the nylon thread when it is extended to a full 6 cm. If the filament has to be shortened to 4 cm or less, the corneal sensation is inferred to have decreased. A value below 2 cm is diagnostic of significant hypaesthesia. Recent methods use mechanoelectric devices to deliver forces of preset duration, intensity, and speed to the cornea for standardized quantifica­tion.


1. Tear Film Abnormalities

A scientific approach to the diagnosis of corneal diseases is incomplete without the evaluation of tear film status. There is no reliable objective test to render a firm diagnosis of dry eyes. Three clinical diagnostic tests in common usage are the Schirmer test, the measurement of tear break-up time, and the rose bengal staining. There are a number of other laboratory tests, which are basically used for research purposes, but not readily available in a clinical setting, such as tear osmolality, tear lysozyme measurements, tear lactoferrin measurement, tear mucin measurement, and goblet cell counts. Currently, meibomian gland dysfunction as in chronic blepharitis and meibomitis is being implicated as an important, but unrecognized cause of dry eye. The test designed to confirm meibomian gland dysfunction is the infrared meibography [Figure - 11][Figure - 12]. The tests are briefly summarized in [Table - 2]

2. Inflammations and Infections

Laboratory tests eminently contribute to the diagnosis of corneal inflammatory and infective disorders. Specific confirmatory cytological and microbiological techniques are applied after making a presumptive clinical diagnosis. Kimura and Thygeson have established the value of cytodiagnosis. The significance of each cell type is listed in [Table - 3]. Specific laboratory investigations for presumptively diagnosed clinical conditions are summarized in [Table - 4].

The technique of obtaining material for microbiological investigations is of extreme importance. Specimen should ideally be obtained before institution of antimicrobial therapy, which may not be possible in case of referred patients who may already be on some treatment. The first step is to obtain a culture from the lids and conjunctiva of both the infected and uninfected eyes using sterile cotton swabs, and preferably calcium alginate swabs moistened with nutrient broth. For obtaining material from the corneal ulcer, under direct visualization with adequate magnification (slit-lamp, operating microscope, or a loupe) and under topical anaesthesia, a Kimura spatula or the bent tip of a 21-gauge needle is used to gently scrape the base and the leading edge of the ulcer. The specimen is used for preparing a smear or a wet-mount and for direct inoculation onto the culture medium. The standard culture media used are the soybean-casein digest broth for saturation of swabs; blood agar plate for aerobic and facultative anaerobic bacteria; chocolate agar for aerobic and facultative anaerobic bacteria, neisseria, and haemophillus; Supplemented thioglycollate broth without indicator for aerobic and anaerobic bacteria; Lowenstein-Jensen agar for mycobacterium and nocardia; Sabouraud's agar with chloramphenicol or gentamicin for fungi; and nonnutrient agar with E. coli for Acanthamoeba.

  Conclusion Top

We have tried to explore the invasive and noninvasive modalities available to diagnose, monitor, and further our basic understanding of diseases of the cornea. A skilled user of these tests must know more than their basic application. A thorough clinical and practical knowledge of the corneal diseases is mandatory for judicious use of the mind-boggling array of investigative procedures currently available, and for drawing conclusive diagnostic support in favour of the clinical presumption.


  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8], [Figure - 9], [Figure - 10], [Figure - 11], [Figure - 12]

  [Table - 1], [Table - 2], [Table - 3], [Table - 4]


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