Patient Selection

The design was a prospective case-control observational study and adhered to the tenets of the Declaration of Helsinki. The study protocol was approved by the Review Board of the Department of Medicine and Ageing Sciences (University of Chieti, Italy). Informed, written consent for research was obtained from all patients prior to enrollment. Twenty eyes of 17 consecutive patients with a clinical suspected diagnosis of total or partial limbal stem cell deficiency based on history, suspect of conjunctivalization of the cornea characterized by late fluorescein staining, neovascularization, and scarring of the cornea were enrolled.

Underlying causes for limbal stem cell deficiency were mucous membrane pemphigoid (n = 4), previous corneal graft failure with chronic ocular surface inflammation (n = 3), severe dry eye in Sjögren syndrome (n = 2), recurrent stromal herpetic keratitis (n = 3), alkali burn of the ocular surface (n = 3), previous limbal stem cell transplantation performed for chemical burn (n = 1), and aniridia (n = 1). There were 7 male and 10 female patients, aged between 11 and 85 years (mean 58.7 ± 19.5).

Ten randomly selected eyes of 10 healthy subjects (5 male and 5 female), aged between 20 and 70 years (mean 54.6 ± 9.3), with no history of ocular surface disease, corneal surgery, or contact lens use were also examined and served as a control group.

A detailed history was taken in every case to ascertain the injury or disease leading to limbal stem cell deficiency. Detailed clinical examination with slit-lamp biomicroscopy was carried out for signs of limbal stem cell deficiency. These signs included loss of normal limbal anatomy; conjunctivalization of the cornea with conjunctival/metaplastic cells that could be highlighted with fluorescein stain; the corresponding corneal surface (epithelium) being thinner and irregular compared to normal corneal epithelium; superficial and/or deep vascularization in the conjunctivalized cornea; and associated ocular surface inflammation. The demographic and clinical data of patients with limbal stem cell deficiency are summarized in Table 1 .

In Vivo Confocal Microscopy

All patients underwent laser scanning in vivo confocal microscopy examination of the limbus and of the corneal surface by 1 experienced examiner (M.L.). In vivo confocal microscopy scans were performed using a digital corneal confocal laser-scanning microscope (HRT II Rostock Cornea Module, diode-laser 670 nm; Heidelberg Engineering GmbH, Heidelberg, Germany). The confocal laser scanning device was equipped with a water immersion objective (Zeiss, Jena, Germany; 63×/N.A. 0.95 W) and permitted an automatic z-scan determination of depth of focus within the cornea. Thus high-contrast digital images with a field of view of 300 × 300 μm were acquired of all corneal layers. The theoretical confocal section thickness is approximately 10 μm. This is the slice thickness (voxel), which is imaged by the confocal microscope to form a 2-dimensional pixel-based digital image. The lateral and transverse resolution is 4 μm.

In vivo confocal microscopy was carried out under topical anesthesia with 0.4% oxybuprocaine. Proper alignment and positioning of the head was maintained with the help of a dedicated movable-target red fixation light for the contralateral eye. A digital camera mounted on a side arm provided a lateral view of the eye and objective lens to monitor the position of the objective lens on the surface of the eye. A drop of 0.2% polyacrylic gel served as coupling medium between the poly(methyl methacrylate) contact cap of the objective lens and cornea (contact lens). In vivo confocal microscopy examination was performed in the central and paracentral cornea, and in 8 clock-hour positions of the limbal area (12, 6, 3, 9, corresponding to superior, inferior, nasal, or temporal limbus; and intermediate positions between the above points of the limbal circumference), using a previously described method.

Sequential images derived from automatic scans and manual frame acquisition throughout the area of interest were obtained with emphasis on visualizing pathologic and nontransparent superficial tissue of the corneal surface. Particular attention was paid in order to obtain a sufficiently large mapping area, which was not limited to the central zone. This was done by moving the objective lens throughout the central and paracentral cornea. At least 60 images from the corneal area and 60 from the limbal area were obtained for each eye, with a total acquisition time of less than 5 minutes for each patient.

For corneal scans the epithelial stratification and cellular morphology were evaluated in the central and paracentral cornea and, in cases of suspected partial limbal stem cell deficiency, also in the areas of epithelial abnormalities detected by slit-lamp biomicroscopic examination, as described above. Particular emphasis was used to analyze epithelial cell morphology, presence of goblet cells, subepithelial vascularization, and subepithelial fibrous tissue. Normal corneal epithelium was defined as multilayered epithelium with specific morphologic characteristics: polygonal and flat cells with hyperreflective nuclei in the superficial layer that progressively decreased in size in the intermediate layers and small cells without detectable nuclei with reflective borders in the basal layer. Conjunctival epithelium was defined as a stratified epithelium with higher reflectivity as compared to corneal epithelium, composed of cells of cuboidal or polygonal morphology, hyporeflective cytoplasm with or without detectable nuclei, and barely defined borders. Round or oval cells with a very reflective homogeneous cytoplasm, interspersed between epithelial cells, were interpreted as goblet cells.

In limbal scans, the presence of palisades of Vogt and a progressive morphologic transition of epithelial cells from the conjunctival to the corneal phenotype in the peripheral cornea adjacent to the limbus were considered an indicator of normal anatomy.

Palisades of Vogt and limbal epithelial transition were considered present when revealed in at least 3 limbal quadrants examined, partially present if revealed in only 1 or 2 sectors, and absent if not detected at all.

Impression Cytology

All patients underwent impression cytology of the central corneal surface after in vivo confocal microscopy examination and specimens were stained with periodic acid–Schiff (PAS) reagents and immunofluorescence staining using antibodies against cytokeratin 12 (K12, corneal-specific) and cytokeratin 19 (K19, conjunctival-specific) to determine the phenotype of cells populating the corneal surface.

Samples with cells covering more than 80% of the membrane area, or samples covering between 50% and 80% where the cells were confluent and present in a defined area of the membrane (not scattered), were considered suitable for diagnostic purposes. For impression cytology the Millicell-CM 0.4 μm (Millipore, Bedford, Massachusetts, USA) membrane was used and the cells were fixed with cytology fixative (Bio-fix; Bio Optica, Milan, Italy).

Samples were stained with PAS reagents (Sigma-Aldrich, St. Louis, Missouri, USA) to identify goblet cells. Goblet cells were counted in 3 randomly selected microscopic fields (×40) by the same masked observer.

For K12 and K19 immunofluorescence staining, the Millicell membranes were hydrated with distilled water and placed in 80% alcohol for 2 minutes. The membranes were washed in distilled water and phosphate-buffered saline (PBS) was added for 2 minutes, followed by 2 washes with Wash Buffer (Dako, Glostrup, Denmark) of 2 minutes each. Then the filters were incubated with ribonuclease A (Sigma-Aldrich) diluted 1:290 in PBS for 20 minutes at room temperature. The specimens were washed and PBS–bovine serum albumin 1% was added for 1 hour at room temperature. Finally, K12 antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) 1:50 or K19 (Santa Cruz Biotechnology) 1:20, both diluted in antibody diluent (Dako), were incubated overnight at 4 C. Samples were washed and anti-goat Alexa fluor 488 (Invitrogen, San Giuliano Milanese, Italy) for K12 or anti-rabbit Alexa Fluor 488 (Invitrogen) for K19, diluted 1:200, and propidium iodide at 1:150 (both in antibody diluent) (Dako), were added and incubated for 1 hour at room temperature. Membranes were mounted with a drop of Fluorescent Mounting Medium (Dako) and the cells visualized with a Zeiss confocal laser scanning microscope (510; Carl Zeiss MicroImaging, GmbH, Vertrieb, Germany). Five different fields for each sample were evaluated and 2 independent masked observers counted the positive-staining cells. K12 positivity and K19 negativity indicated corneal epithelium, while K12 negativity and K19 positivity indicated presence of conjunctival epithelium. The presence of both K12 and K19 positivity in different areas of the corneal surface was interpreted as mixed epithelium. All evaluations of impression cytology specimens were performed by 2 independent observers masked to the details of the staining technique used.

The diagnosis of total limbal stem cell deficiency was considered when impression cytology samples were negative for K12, positive for K19 with or without PAS staining evidence of goblet cells, and in vivo confocal microscopy showed the presence of conjunctival epithelial morphology. Conversely, the diagnosis of partial limbal stem cell deficiency was made when impression cytology (positivity of both K12 and K19 with or without goblet cells) and in vivo confocal microscopy showed the presence of both corneal and conjunctival epithelial phenotype onto the corneal surface, distributed in various patterns (mosaic or sectorial).

Statistical Analysis

To assess the inter-method agreement, Cohen’s kappa coefficient (κ) was determined in order to evaluate the degree of agreement between categorical data represented by the 3 diagnostic possibilities assessed by impression cytology and in vivo confocal microscopy on the corneal surface: conjunctival epithelium, corneal epithelium, and mixed epithelium (presence of both types of epithelia). The κ coefficient (ranging from 0-1) describes the strength of agreement between methods (0.00 to 0.40 = poor to fair; 0.41 to 0.80 = moderate to substantial; 0.81 to 1.00 = high to perfect agreement). Statistical analysis was performed with SPSS 11.5 for Windows (SPSS Inc, Chicago, Ilinois, USA).

Corneal Impression Cytology and In Vivo Confocal Microscopy

The results are summarized in Table 2 . Overall, in vivo confocal microscopy and impression cytology agreement in testing the diagnostic hypotheses was high (κ = 0.85). The 2 methods were concordant in 18 out of 20 examined eyes (90%). In vivo confocal microscopy and impression cytology concordantly revealed the presence of just corneal epithelium in 7 cases ( Figure 1 ), just conjunctival epithelium (total limbal stem cell deficiency) in 5 cases ( Figure 2 ), and both types of epithelia in a variable proportion (defined as “mixed epithelium”) in 6 cases (partial limbal stem cell deficiency, Figure 3 ).

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