MICROSCOPY AND CLINICAL APPLICATIONS

An impression cytology usually removes only 13 cell layers and does not yield the same information as a flat mount or cross section preparation of the ocular surface. It is therefore ideal for studying the surface epithelium rather than the basal epithelium or the basement membrane (fig 2A). However, using multiple impressions of the same area (in vivo or in vitro cadaver eye) we were able to demonstrate the morphology of the basal limbal epithelium. The limbal cells are smaller, more densely packed, and have a greater nucleus to cytoplasm ratio compared to adjacent corneal and conjunctival cells (fig 2B). Morphologically Egbert first used this method to determine the density of goblet cells in different areas of the conjunctiva.1 He found the greatest density of goblet cells in the nasal palpebral conjunctiva, with decreasing densities in the temporal and palpebral conjunctiva, bulbar conjunctiva near the fornices, and the bulbar conjunctiva near the limbus. His results were similar to those obtained by studies of whole mounts of conjunctiva. Adams studied the morphology of normal human conjunctival mucus using impression cytology and described granules, strands, and structureless patterns of mucus on different parts of the conjunctiva.8

Figure 2  (A) Impression cytology of normal corneal surface showing corneal epithelial cells. Normal cells are flat with a prominent nucleus. The nuclear cytoplasmic ratio is low (x100, periodic acid Schiff staining). (B) Impression cytology of normal transition zone from cornea to limbus (x40, periodic acid Schiff staining). The limbal epithelial cells are small, densely packed with a high nuclear cytoplasmic ratio. The limbal zone is clearly demarcated from the adjacent corneal epithelial cells.

Impression cytology has also been used in the evaluation of ocular surface diseases such as keratoconjunctivitis sicca,9,10 vitamin A deficiency,11 cicatricial pemphigoid,12 atopic disease,13 superior limbic keratoconjunctivitis and mucopolysaccharidoses,3 vernal keratoconjunctivitis,14 and the effect on these of various therapies. Tseng classified conjunctival squamous metaplasia into six stages according to the presence or absence of goblet cells and goblet cell density, morphological changes of the nucleus, nucleus-cytoplasm ratios, metachromatic changes of cytoplasmic colour, and emergence of keratinisation.2 Nelson graded conjunctival impression cytology specimens (grades 03, table 1) based on the appearance of the epithelial cells and the numbers of goblet cells.15 A specimen of small, round epithelial cells with large nuclei and more than 500 goblet cells/mm2 was considered grade 0, whereas another of large polygonal epithelial cells with small nuclei and less than 100 goblet cells/mm2 was considered grade 3. All specimens that were grade 2 or more were abnormal. The findings of grades 2 and 3 on the interpalpebral conjunctiva and grades 0 and 1 on the inferior palpebral ocular surface in the absence of inflammatory cells suggest a diagnosis of keratoconjunctivitis sicca, whereas grades 2 and 3 on both the bulbar and palpebral conjunctiva suggest an intrinsic ocular surface disease such as ocular cicatricial pemphigoid, Stevens-Johnson syndrome, or severe chemical burns.15 The presence of inflammatory cells suggests that the disease is active. Marner studied impression cytology specimens of conjunctival epithelial cells of patients with keratoconjunctivitis sicca and described a snake-like appearance of nuclear chromatin in clusters of abnormal cells from the upper bulbar conjunctiva (fig 3).9 Prabhasawat and Tseng used impression cytology to demonstrate normal conjunctival epithelial cells and goblet cells following ocular surface reconstruction by preserved amniotic membrane.16 Modifications of the impression cytology technique were made to study cytokeratin expression in bulbar conjunctiva by using pure nitrocellulose membranes and immunocytochemical staining.4 The conjunctiva was found to demonstrate a unique cytokeratin expression pattern containing cytokeratins characteristic of non-keratinised, stratified epithelia (K4 and K13) as well as others more typical of a simple differentiation pattern (K8 and K19), a glandular differentiation pattern (K7), or both.4 This technique was later used in the preoperative diagnosis of seborrhoeic keratosis of the conjunctiva simulating a malignant melanoma.17 Thiel et al used a biopore membrane device to collect ocular surface specimens and apply immunopathological methods to diagnose superficial viral infections like herpes simplex virus, varicella zoster virus, and adenovirus.5

Table 1  Nelson’s classification for squamous metaplasia

Figure 3  Impression cytology of the conjunctival surface showing snake-like chromatin in keratoconjunctivits sicca (x100, periodic acid Schiff staining).

More recently impression cytology has been used to demonstrate conjunctival metaplasia as a result of the use of topical antiglaucoma drugs.18,19 Free radical production was found in patients on long term antiglaucoma treatment and contact lens wearers by investigations on impression cytology specimens.20 Immunohistochemistry on impression cytology specimens has been used to compare the efficacy of drugs and to determine the mechanism of action of topical agents in vernal conjunctivitis.21 Impression cytology has also helped in evaluation of ocular surface changes after excimer laser phototherapeutic keratectomy in patients with corneal dystrophies and other corneal pathology.22 It has enabled documentation of limbal cell deficiency in patients with ocular burns and other surface disorders by demonstrating the presence of goblet cells on the corneal surface (fig 4).23 Limbal stem cell deficiency has also been assessed using immunoperoxidase staining for cytokeratins: K3 for corneal, and K 19 for conjunctival phenotype. The limitation to this method was inadequate sampling of cells in 58% of cases (defined as less than 50% cellularity in specimen). Western blotting has been used to demonstrate increased expression of epidermal growth factor receptors ErbB2 and ErbB3 in patients with keratoconjunctivitis sicca.24

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