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The technique of impression cytology was established by Egbert et al in 1977 for studying goblet cells. The basic principle of impression cytology is the application of cellulose acetate filter paper to the ocular surface for the collection of superficial layers lining the ocular surface following which histological, immunohistological, or molecular analysis of the cells can be done. Impression cytology is a very useful, relatively non-invasive tool for assessing ocular surface in various dry eye disorders, such as keratoconjunctivitis sicca (KCS), cicatricial ocular pemphigoid, and vitamin A deficiency.
|Ocular surface disorders||Neoplasia|
|Keratoconjunctivitis sicca||Conjunctival squamous metaplasia|
|Atopic keratoconjunctivitis||Ocular surface squamous neoplasia diagnosis and followup|
|Allergic rhinoconjunctivitis||Conjunctival melanosis|
|Dry eye syndrome|
|Vitamin A deficiency|
|Stem cell deficiency|
|Ocualr Cicatricial pemphigoid|
|Superior limbic keratoconjunctivits|
|Ocular surface changes after excimer laser phototherapeutic keratectomy in patients with corneal dystrophy|
|Demonstration of cyst and trophozoite of acanthamoeba.|
|Assists diagnosis of viral and chlamydial infections|
|Demonstration of conjunctival changes in cystic fibrosis|
|Contact lens wearing status|
Impression cytology is used for diagnosing a large number of ocular surface disorders and neoplasia. The various indications of impression cytology are summarized in Table 1.
Conjunctiva is a fine, translucent mucous membrane that covers the anterior surface of the globe and posterior side of the eyelids and is continuous with the corneal epithelium. Conjunctival goblet cells are found throughout the conjunctiva, residing between the epithelial cells, arising from the basal layer of the epithelium, where they gradually enlarge as they reach the surface. These mucin secreting goblet cells are more concentrated in the inferonasal quadrant. Chronic inflammation and severe dry eyes cause destruction of goblet cells; and in cases of limbal stem cell deficiency, there is conjunctivalisation of the cornea, where goblet cells can be seen on the corneal surface.
Technique of impression cytology
Impression cytology is performed under topical anesthesia (eg-proparacaine 0.5%) although it can be performed without topical anesthesia as well. It is critical to dry the area of interest as much as possible to maximize yield. Therefore, excessive tears or medications should be wiped away from the area to be sampled.
Filter paper Preparation
The type of filter paper used should be selected based on the purpose of the study. Yield increases with larger pores but cell detail is diminished. On the other hand, yield diminishes with smaller pores but cell detail is better preserved. As such, cellulose acetate filter paper/millimeter filter paper with a pore size 0.22 mm is often employed since it renders the best results. Filter paper is typically trimmed into 5mm strips with one end square and the other end tapered. The surface of the paper is marked before applying on the ocular surface for orientation. The paper removes mucous secretions as well as the sheets of epithelial cells and goblet cells from the conjunctival surface.
Egbert et al used Millipore filters to collect conjunctival specimens, which were then air-dried and stained with periodic acid Schiff (PAS) and hematoxylin. Tseng subsequently modified this technique, employing 5mm cellulose acetate filter paper for sample collection. After instilling a topical anesthetic agent, the marked filter paper strip is gently pressed with a glass rod or tip of a Goldmann applanator over the area to be sampled for 5-10 sec. During the period of contact, it is important that the lids are held away from the paper, and filter paper is not allowed to be wetted by tear fluid. If the paper gets wet, the yield of cells will be poor.
Preparation of staining solutions
Staining solutions used in impression cytology include Gill’s hematoxylin, modified OG-6, Scott’s tap water substitute.
Gill’s hematoxylin  is prepared by combining 365 ml of distilled water, 125 ml of ethylene glycol, 1 g of anhydrous hematoxylin, 0.1 g of sodium iodate, 8.8 g of aluminum sulfate, and 10 ml of glacial acetic acid. The chemicals are stirred for 1 hour on a magnetic mixer at room temperature and the final solution is filtered through Whatman No 1 filter paper before using it for the first time. Scott’s tap water substitute consists of 1 g sodium bicarbonate and 5 g magnesium sulfate, anhydrous or 10 g magnesium sulfate, crystalline in 500 ml of tap water. The pH of this solution is 8.02. Modified orange G is made of 10 ml orange G, 10% total dye content (TDC) aqueous solution combined with 490 ml of 95% ethyl alcohol and 0.075 g phosphotungstic acid.
Various staining techniques have been delineated depending on the solutions used. A simplified description of the staining technique most commonly practiced is described below: The sample is immediately transferred into a Coplin jar containing 95% ethyl alcohol for fixation. Fixation is necessary to inhibit autolysis and bacterial contamination and to provide conditions that will enhance the effects of the various biological dyes. Filter paper is then clipped onto to glass slide keeping the marked surface upward for staining. The slide is then dipped in hematoxylin stain followed by eosin stain for 30 seconds. Hematoxylin stains cell nuclei a blue-black color, allowing for microscopic examination. Dehydration is done using butyl alcohol. Clearing is then done in Coplin jar with an equal amount of butyl alcohol and xylene. The slide is then left overnight in xylene to make the filter paper transparent.
Special techniques have been devised for studying specimens by electron microscopy in which the specimen on cellulose acetate paper is fixed in 4% phosphate-buffered formaldehyde with 1% glutaraldehyde and ruthenium red dye and is postfixed in buffered osmium fixative. It is dehydrated and embedded in resin. For immunohistochemical staining, the specimen is collected on a pure nitrocellulose membrane, then fixed with a spray fixative, and transferred onto a poly-L-lysine coated glass slide, and dried. The slide is subsequently placed in acetone for 1 hour with continuous agitation to dissolve the filter membrane. The slide is then washed for 5 minutes with tap water, and subjected to cellulose digestion for 2 hours at 37°C to remove residual membrane material before proceeding to immunocytochemical staining. Xylene for digestion is avoided since it causes destruction of all cell surface antigens. Modifications in this technique help to study cytokeratin expression in the bulbar conjunctiva for instance.
Interpretation: Normal cells are flat with a prominent nucleus with a low nuclear-cytoplasmic ratio. Limbal epithelial cells are smaller, more densely packed, and have a higher nucleus-cytoplasmic ratio.
A decrease in goblet cells in both palpebral and bulbar conjunctiva is suggestive of intrinsic ocular surface disease like ocular cicatricial pemphigoid, Stevens-Johnson syndrome, or severe chemical burns. The presence of inflammatory cells is suggestive of active inflammation.
Advantages of impression cytology are as follows:
- Ability to diagnose ocular surface disorders
- Simple to perform and non-invasive
- Causes minimal discomfort to the patient
- Multiple samples can be obtained in one sitting
Limitations of impression cytology are:
- May be a time-consuming and cumbersome procedure
- Not yet a routine diagnostic tool
- Requires expert ocular pathologist, microbiologist, and ophthalmologist
- Cannot differentiate between dysplasia and invasive squamous cell carcinoma
Impression cytology has 80% accuracy for predicting the histological diagnosis of OSSN and has 77% predictability rate for moderate dysplasia. In acanthamoeba keratitis, the yield can be as high as 94.6%.
- Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy. Am J Ophthalmol 1977;84:798–801
- Dogru M, Katakami C, Nakagawa N, Tetsumoto K, Yamamoto M Impression cytology in atopic dermatitis Ophthalmology. 1998 Aug; 105(8):1478-84.
- Tseng SCG. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology 1985;92:728–33.
- Gill GW, Frost JK, Miller KA A new formula for a half-oxidized hematoxylin solution that neither overstains nor requires differentiation. Acta Cytol. 1974 Jul-Aug; 18(4):300-11.
- Maskin SL, Bodé DD Electron microscopy of impression-acquired conjunctival epithelial cells.Ophthalmology. 1986 Dec; 93(12):1518-23.
- Krenzer KL, Freddo TF Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology.Invest Ophthalmol Vis Sci. 1997 Jan; 38(1):142-52.
- Nelson JD, Wright JC. Conjunctival goblet cell densities in ocular surface disease. Arch Ophthalmol 1984;102:1049–51.
- Tole DM, McKelvie PA, Daniell M. Reliability of impression cytology for the diagnosis of ocular surface squamous neoplasia employing the bio pore membrane. Br J Ophthalmol. 2001 Feb;85(2):154-8.
- Nolan GR, Hirst LW, Wright RJ, et al. Application of impression cytology to the diagnosis of conjunctival neoplasms. Diagn Cytopathol. 1994;11(30:246-9.