Impression Cytology

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Impression cytology


The technique of impression cytology was established by Egbert et al in 1977 for studying goblet cells.[1] The basic principle of impression cytology is application of cellulose acetate filter paper to the ocular surface for 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 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


·     Keratoconjunctivitis sicca

·      Atopic keratoconjunctivitis

·      Allergic rhinoconjunctivitis

·         Dry eye syndrome

·         Vitamin A deficiency

·         Stem cell deficiency

·         Ocualr Cicatricial pemphigoid

·         Superior limbic keratoconjunctivits

·        Mucopolysaccharidoses

·         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

·        Filtering bleb

·        Contact lens wearing status

·   Conjunctival sqaumous metaplasia

·   Ocular surface squamous neoplasia diagnosis and followup

·   Conjunctival melanosis.

Indications of impression cytology Table 1

Impression cytology is used for diagnosing a large number of ocular surface disorders and neoplasia.The various indications of impression cytology are summarized in table1.[2]

Relevant anatomy

Conjunctiva is a fine, translucent mucous membrane which covers the anterior surface of eyeball and posterior side of the eyelids and then becomes continuous with the corneal epithelium. Conjunctival goblet cells are found throughout conjunctiva lying in between cells of epithelium and arise from basal layer of epithelium they gradually enlarge and become larger as they reach the surface. These mucin secreting goblet cells are more concentrated inferonasal . Chronic inflammation and severe dry eyes causes destruction of goblet cells and in cases of limbal stem cell deficiency there is conjunctivalisation of cornea and goblet cells can be demonstrated on the corneal surface.

Technique of impression cytology

Patient Preparation

Impression cytology is performed under topical anaesthesia (eg-proparacaine 0.5%) although can also be performed without topical anaesthesia. Excessive tears or medications should be wiped away from the area to be sampled.

Filter paper Preparation

Cellulose acetate filter paper/millimeter filter paper with pore size 0.22 mm is used it removes mucous secretions as well as the sheets of epithelial cells and goblet cells from the conjunctival surface. Filter paper is trimmed into 5mm strips with one end square and other end tapering. Surface of paper is marked before applying on the ocular surface .

Specimen collection

Egbert et al used Millipore filters to collect conjunctival specimens, which were then air dried and stained with periodic acid Schiff (PAS) and haematoxylin, Tseng[3] modified this technique and used 5mm cellulose acetate filter paper for sample collection. After instilling topical anaesthetic agent the marked filter paper strip is gently pressed with glass rod on 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 as if the paper gets wet, unduely athe yield of cells will be poor.

Preparation of staining solutions

Staining solutions used in impression cytology include Gill’s haematoxylin, modified OG-6,Scott’s tap water substitute.

Gill’s haematoxylin [4] is prepared by combining 365 ml of distilled water, 125 ml of ethylene glycol, 1 g of anhydrous haematoxylin, 0.1 g of sodium iodate, 8.8 g of aluminium sulphate, 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.

Staining technique

Various staining techniques are followed depending on the solutions used. A simplified description of staining technique most commonly practiced is described below: The sample is immediately transferred into 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 various biological dyes Filter paper is then clipped on to glass slide keeping marked surface upward for staining . Firstly slide with filter paper is dipped in haematoxylin stain followed by eosin stain for 30 seconds.Hematoxylin stains cell nuclei a blue-black color resulting in enhanced observation of the epithelial cells. Dehydration is done using butyl alcohol Clearing is then done in capolin jar with equal amount of butyl alcohol and xylene The slide is then left overnight in xylene for transparency of filter paper.

Special Techniques

Special techniques have been devised for studying the 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 post fixed in buffered osmium fixative,it is dehydrated and embedded in resin.[5] For immunohistochemical staining specimen is collected on a pure nitrocellulose membrane which was then fixed with a spray fixative and transferred on to a poly-L-lysine coated glass slide, and dried. The slide is then placed in acetone for 1 hour with continuous agitation to dissolve the filter membrane slide is then washed for 5 minutes in tap water, and subjected to cellulose digestion for 2 hours at 37°C to remove residual membrane material before proceeding to immunocytochemical staining,this is done so because using xylene for digestion causes destruction of all cell surface antigens.[6] Interpretation:Normal cells are flat with a prominent nucleus and have a low nuclear cytoplasmic ratio.Limbal epithelial cells are samller,densely packed and have a higher nucleus cytoplasmic ratio. 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.Presence of inflammatory cells is suggestive of active inflammation. Modifications in the impression cytology technique helps to study cytokeratin expression in bulbar conjunctiva .


The various advantages of impression cytology are as follows:

  • Impression cytology is an ideal method for diagnosing ocular surface disorder
  • Simple to perform and non invasive
  • Causes minimal discomfort to patient
  • Multiple samples can be obtained in one sitting.


Limitations of impression cytology are:

  • Impression cytology is time consuming and cumbersome procedure
  • It is not yet a routine diagnostic tool
  • Impression cytology 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 [8] and has 77% predictability rate for moderate dysplasia.[9] In acanthamoeba keratitis the yield can be as high as 94.6%.[10]


1) Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy. Am J Ophthalmol 1977;84:798–801

2) Dogru M, Katakami C, Nakagawa N, Tetsumoto K, Yamamoto M Impression cytology in atopic dermatitis Ophthalmology. 1998 Aug; 105(8):1478-84.

3) Tseng SCG. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology 1985;92:728–33.

4) 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.

5) Maskin SL, Bodé DD Electron microscopy of impression-acquired conjunctival epithelial cells.Ophthalmology. 1986 Dec; 93(12):1518-23.

6) 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.

7) Nelson JD, Wright JC. Conjunctival goblet cell densities in ocular surface disease. Arch Ophthalmol 1984;102:1049–51.

8) 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.

9) 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.

10) Kanavi MR, Hosseini B, Javadi M, Rakhshani N, Javadi MA. Impression cytology in eyes with clinical and confocal scan features of acanthamoeba keratitis. J Ophthalmic Vis Res. 2013 Jul;8(3):207-12.