Figure 4  Impression cytology of the conjunctivalised corneal surface in limbal stem cell deficiency showing reddish pink goblet cells (x100, periodic acid Schiff staining).

Cells harvested from the cornea by impression cytology have been used for investigation of DNA polymorphisms using the polymerase chain reaction (fig 5).25 MUC 7 gene has been demonstrated in conjunctival cells obtained by impression cytology detecting elevated mRNA levels using semi-quantitative reverse transcriptase polymerase chain reaction (RT PCR).26 RT-PCR techniques have been used to show that defensin h-BD2 is preferentially expressed in moderate dry eye patients, and demonstrated that it can be induced in cultured conjunctival cells by exposing them to inflammatory cytokines.27

Figure 5  rtPCR image of cDNA extracted from cells obtained by impression cytology from cornea (lanes 3, 5, 7) and conjunctiva (lanes 2, 4, 6). Primers for human ? defensins 1 and 2 were used. (Reproduced by courtesy of Masahel Al-Abed, Larry A Donoso Laboratory for Eye Research, Division of Ophthalmology, University of Nottingham).

Pisella et al have used flow cytometry on conjunctival impression cytology specimens in dry eye and acne rosacea patients and found upregulation of HLA DR and ICAM-1 expression compared to control samples, supporting an inflammatory basis for their ocular surface disease.28 They also found a reduction in M-1 positive staining cells (goblet cell stain), and suggested inflammation may be responsible. Cytokines IL-6, IL-8, IL-10 have been found to be elevated in patients on chronic antiglaucoma medications using flow cytometry.29

Impression cytology has been used widely as a non-invasive method for conjunctival biopsy for suspected ocular surface squamous neoplasia (OSSN).30,31 Using biopore membrane for specimen collection an 80% correlation was found between impression cytology diagnosis and histopathology specimens obtained from incisional biopsy (fig 6). Keratinising malignancies offer the highest chance of false negatives because of paucity of cells in the specimen and should be kept in mind in such cases. Detailed cytomorphology of OSSN using impression cytology has been described.32 Mitomycin C (MMC) has gained acceptance for the treatment of OSSN especially in cases of recurrence or extensive disease where excision may jeopardise limbal stem cell function. McKelvie et al have followed patients after treatment with MMC for OSSN and, using impression cytology, demonstrated eradication of malignant cells, primarily by apoptosis, and a small amount of necrosis accompanied by inflammatory cells.33 Normal cells undergo cytoplasmic enlargement and vacuolisation, and nuclear enlargement, but maintained a normal nuclear to cytoplasmic ratio. These changes persisted for a variable time following treatment, but resolved eventually. Impression cytology has been studied in pigmented conjunctival lesions and predicted conjunctival melanocytic malignancy in 73% of cases studied.34

Figure 6  Impression cytology of the ocular surface showing dysplastic squamous cells with increased nucleus:cytoplasmic ratio, hyperchromatic nuclei, irregular nuclear membranes, and prominent nucleoli (x250 magnification, Papanicolaou stain). (Reproduced by courtesy of Dr Ushma Samaraweera, Department of Pathology, Prince of Wales Hospital, Sydney, Australia).

Impression cytology has also been used to diagnose acanthomoeba keratitis in three patients by visualisation of cysts and trophozoites taken from the superficial cornea from patients with clinically suspicious infections.35

While there are numerous clinical and research applications of impression cytology, it has not yet become a routine diagnostic tool in most clinics because it is relatively cumbersome and time consuming for both the clinician and pathologist. However, the ability to obtain multiple samples of the ocular surface at one sitting with minimal discomfort to the patient makes it an ideal method of investigating ocular surface disorders when the diagnosis is not clinically obvious or when the clinical diagnosis needs to be substantiated and documented. It is also a handy research tool. We recommend that major ophthalmic centres should develop and introduce this technique into routine clinical practice. For this to be achieved a team approach including the ophthalmologist, pathologist, microbiologist, and the immunologist is essential.

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