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Year : 2017  |  Volume : 55  |  Issue : 4  |  Page : 267-272

Correlation of impression cytology with histopathology in the diagnosis of ocular surface squamous neoplasia

1 Department of Cornea, RIOGOH, Chennai, Tamil Nadu, India
2 The Tamil Nadu Dr. M.G.R. Medical University, Chennai, Tamil Nadu, India

Date of Web Publication25-Apr-2018

Correspondence Address:
Dr. Sharmiladevi Vinod
Department of Cornea, RIOGOH, Egmore, Chennai, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tjosr.tjosr_45_17

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Background: Ocular surface squamous neoplasia (OSSN) includes a wide spectrum of conjunctival and corneal intraepithelial neoplasia which can manifest as dysplasia, carcinoma in situ, and invasive squamous cell carcinoma. OSSN can be diagnosed by certain noninvasive techniques as well as invasive histopathological examination. One of such noninvasive techniques is impression cytology which refers to the histological study of superficial layers of ocular surface epithelium. Aim: The aim of this study was to assess the accuracy of impression cytology in the diagnosis of OSSN using cellulose acetate paper by correlating with histopathology. Materials and Methods: This is a prospective observational and interventional study conducted at a tertiary referral center in Chennai. This study was conducted from January 2016 to July 2017 and included 43 eyes of 42 patients presenting with conjunctival mass at the limbus or over the conjunctiva. Hospital ethics committee approval was obtained. Results: A total of 43 excision biopsies of 42 patients suspected for OSSN were performed. There was correlation in 36 eyes (83.72%), and in 7 eyes, there was no correlation (16.27%). The cases having hyperkeratotic lesions were missed by the impression cytology. Conclusion: Impression cytology can be used as a screening tool in the diagnosis of OSSN and in the follow-up of patients with recurrence. However, histopathology always remains the gold standard in diagnosing OSSN.

Keywords: Dysplasia, histopathology, impression cytology, ocular surface squamous neoplasia, squamous cell carcinoma

How to cite this article:
Vinod S, Priyadharshni R R, Anandababu M, Meenakshi B, Santhosh JM. Correlation of impression cytology with histopathology in the diagnosis of ocular surface squamous neoplasia. TNOA J Ophthalmic Sci Res 2017;55:267-72

How to cite this URL:
Vinod S, Priyadharshni R R, Anandababu M, Meenakshi B, Santhosh JM. Correlation of impression cytology with histopathology in the diagnosis of ocular surface squamous neoplasia. TNOA J Ophthalmic Sci Res [serial online] 2017 [cited 2020 Aug 9];55:267-72. Available from: http://www.tnoajosr.com/text.asp?2017/55/4/267/231131

  Introduction Top

Ocular surface squamous neoplasia (OSSN) includes a wide spectrum of conjunctival and corneal intraepithelial neoplasia. The lesions may vary from mild dysplastic changes to dangerous invasive squamous cell carcinoma (SCC). Early manifestations are small masses at or around the limbus mimicking pterygia, occurring in middle-aged patients.[1] However, their potential for malignancy is quite high, and hence, the early diagnosis assumes greater significance. It was Lee and Hirst who coined the term OSSN. The two major risk factors are ultraviolet-B light and human papillomavirus (HPV). A number of studies have been successful in detecting HPV Type 16 and 18 in OSSN.[1] Other reported risk factors include exposure to petroleum products, heavy cigarette smoking, chemicals such as trifluridine, arsenicals, beryllium, ocular surface injury, Vitamin A deficiency, light pigmentation of hair and eye, defective DNA repair in xeroderma pigmentosum, family origin in the British, Austria, or Switzerland, infection with HIV, and other immunocompromised states.[2]

OSSN occurs in the sun-exposed areas of the conjunctiva at the limbus in elderly individuals. Excessive exposure to ultraviolet light causes DNA damage. Failure of DNA repair leads to the formation of carcinoma. The average age of presentation is usually in the sixth and seventh decades. The p53 gene is a common cellular target in human carcinogenesis and is thought to have an important role in cellular proliferation. OSSN is a slow-growing tumor of low-grade malignancy, which rarely metastasise.[3]

OSSN can be classified as preinvasive lesions and invasive SCC. The preinvasive lesions are again classified as mild, moderate, and severe dysplasia depending on the degree of involvement. Mild dysplasia involves the lower third of the epithelium, moderate dysplasia extends into the middle third, and severe dysplasia refers to the full-thickness involvement. Invasive OSSN or SCC shows nests of neoplastic cells that have penetrated the epithelial basement membrane and spread into the underlying stroma. Tumor cells may be well-differentiated or ill-differentiated. Two types of cells may be seen interspersed along with squamous cells – spindle cells or mucoepidermoid cells. The latter is notorious for intraorbital extension as well as early recurrence if left untreated.

Egbert et al. first introduced impression cytology into ophthalmology in 1977. They used cellulose acetate filter paper.[4] Thiel et al. described the use of Biopore membrane.[5] Hatchell and sommer suggested that impression cytology could be used for early detection of Vitamin A deficiency.[6] Nolan et al. first reported application of impression cytology using acetate filter paper for the diagnosis of conjunctival neoplasia.[7] Tseng has done staging of conjunctival metaplasia by impression cytology in 1985.[8] Tole et al. studied the reliability of impression cytology for the diagnosis of OSSN employing the Biopore membrane. Impression cytology has been found to be useful in assessing the ocular surface in various dry eye disorders such as keratoconjunctivitis sicca, ocular pemphigoid, Vitamin A deficiency, and ocular squamous neoplasia.[9]

  Materials and Methods Top

Inclusion criteria

Patients with elevated conjunctival limbal lesions accompanied by feeder vessels were included in the study.

Exclusion criteria

Patients with extensive involvement, patients with exclusive corneal involvement, and patients with recurrence were excluded from the study.

  Methodology Top

Complete slit-lamp examination was done to describe the extension of the lesion, presence of any feeder vessel, or pigmentation following which all cases were subjected to impression cytology [Figure 1]. In this technique, cellulose acetate filter paper was used and it was pressed firmly against the lesion for about 5–10 s after instilling 4% xylocaine drops. The smear was made on glass slide using filter paper. The slide was fixed with 100% isopropyl alcohol for half an hour. After staining with hematoxylin and eosin, slides were studied under light microscope by a single pathologist. Slides were observed for the presence of dysplastic cells, inflammatory cells, and hyperkeratosis.
Figure 1: Different types of ocular surface squamous neoplasia in our study

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Shield's no touch technique

Tumor resection was performed under local peribulbar or retrobulbar anesthesia, using a 1:1 mixture of 2% lidocaine and 0.75% bupivacaine. Phenylephrine 2.5% drops to induce vasoconstriction. After outlining the borders of the tumor, 4-mm margins are marked with the help of calipers at the scleral edges. The conjunctiva is lifted with forceps, and the first incision was made with a pair of Westcott scissors. Alcohol kerato-epitheliectomy was done on the corneal side. Initial tumor dissection is localized to the marked margins, and any contact of the tumor with the surgical instruments was avoided to stop tumor seeding. After completion of the dissection of the peripheral marked margins, the dissection was directed towards the centre of the tumor. In the event of tumor adherence to the sclera, a partial thickness sclerectomy is performed with a blade and forceps. Following this, double freeze-thaw cryotherapy of the margins was done. Amniotic membrane grafting was done for better healing. Once the lesion is removed en bloc, the specimen was marked in the proper orientation with sutures and then transferred to a piece of pencil-marked cardboard. The specimen was sent to pathology in formalin [Figure 2]. For larger lesions, chemoreduction was done with mitomycin c. [Figure 3] shows Preoperative and postoperative pictures.
Figure 2: Excised tissue with proper marking of borders

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Figure 3: Preoperative and postoperative pictures

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  Results Top

Cytology and histopathology reports were compared. The presence of dysplastic cells in impression cytology and neoplasia in histopathology was considered as correlation. 42 patients were included in this study. Out of 42 patients, 23 (54.76%) were males and the remaining 19 (45.23%) were females [Figure 4].
Figure 4: Pie chart showing sex distribution

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Five patients (11.90%) had SCC. According to morphology, [Figure 5] gelatinous type constitutes 60.46%, leukoplakic – 13.95%, papilliform – 9.30%, and pigmentary – 16.27% [Figure 6].
Figure 5: Bar chart showing different morphological types of ocular surface squamous neoplasia

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Figure 6: Pigmentary ocular surface squamous neoplasia

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One patient (2.38%) had bilateral presentation [Figure 7]. Four patients (9.52%) were found to have HIV infection.
Figure 7: Bilateral presentation of ocular surface squamous neoplasia in a HIV patient

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  Discussion Top

Impression cytology refers to the application of a cellulose acetate filter to the ocular surface to remove the superficial layers of the ocular surface epithelium. These cells can then be subjected to histological, immunohistological, or molecular analysis. Proper technique is essential as the number of cells sampled can vary considerably. In general, two to three layers of cells are removed in one application, but deeper cells can be accessed by repeat application over the same site. Applications for impression cytology include diagnosing a wide range of ocular surface disorders, documenting sequential changes in the conjunctival and corneal surface over time, staging conjunctival squamous metaplasia, and monitoring effects of treatment.

In this study impression, cytology and histopathology had correlation [Figure 8] in 36 eyes [83.72%] [Table 1]. The remaining 7 (16.27%) eyes had no correlation [Figure 9].
Figure 8: Correlation between impression cytology and histopathology

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Table 1: Correlation between impression cytology and histopathology

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Figure 9: Noncorrelation between impression cytology and histopathology

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  • True positive: 35 (ocular surface squamous neoplasia)
  • True negative: 1 (Nevi).


  • False positives: 4 – Out of these, 2 eyes were found to have dysplastic cells in impression cytology but were diagnosed to be schwannoma and melanoma in histopathology [Figure 10]. One eye had inflammatory cells in histopathology, and the other one was nevi
  • False negatives: 3 – However impression cytology failed to diagnose dysplastic cells in the hyperkeratotic lesions.
Figure 10: Schwannoma and melanoma in histopathological examination

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Statistical indices were calculated [Table 2]. The positive predictive value of this cytological test is quite high which was about 89.74%, and the sensitivity was about 92.11%. In our study the accuracy was 83.72% [Table 3] when Compared with other similar studies.
Table 2: Statistical indices

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Table 3: Comparison with other studies

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  Conclusion Top

Impression cytology is a simple, noninvasive technique with a high correlation rate in the diagnosis of OSSN. It can be used as a routine investigation in patients with suspected OSSN. It is especially useful in patients with suspected recurrence following excision biopsy. Impression cytology has reasonably good accuracy. It has a definite role for the follow-up of lesions, after primary treatment, whether surgical or topical chemotherapy. Major ophthalmic centers should develop and introduce this technique into routine clinical practice. This is best achieved with a team approach including the ophthalmologist, pathologist, microbiologist, and the immunologist. However, histopathology remains the gold standard investigation for the diagnosis of OSSN.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Tulvatana W, Bhattarakosol P, Sansopha L, Sipiyarak W, Kowitdamrong E, Paisuntornsug T,et al. Risk factors for conjunctival squamous cell neoplasia: A matched case-control study. Br J Ophthalmol 2003;87:396-8.  Back to cited text no. 1
Mittal R, Rath S, Vemuganti GK. Ocular surface squamous neoplasia – Review of etio-pathogenesis and an update on clinico-pathological diagnosis. Saudi J Ophthalmol 2013;27:177-86.  Back to cited text no. 2
Anil Radhakrishnan MS. Ocular surface squamous neoplasia [OSSN] – A brief review. Kerala J Ophthalmol 2011;xxiii:347-51.  Back to cited text no. 3
Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy. Am J Ophthalmol 1977;84:798-801.  Back to cited text no. 4
Thiel MA, Bossart W, Bernauer W. Improved impression cytology techniques for the immunopathological diagnosis of superficial viral infections. Br J Ophthalmol 1997;81:984-8.  Back to cited text no. 5
Hatchell DL, Sommer A. Detection of ocular surface abnormalities in experimental Vitamin A deficiency. Arch Ophthalmol 1984;102:1389-93.  Back to cited text no. 6
Nolan GR, Hirst LW, Wright RG, Bancroft BJ. Application of impression cytology to the diagnosis of conjunctival neoplasms. Diagn Cytopathol 1994;11:246-9.  Back to cited text no. 7
Tseng SC. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology 1985;92:728-33.  Back to cited text no. 8
Tole DM, McKelvie PA, Daniell M. Reliability of impression cytology for the diagnosis of ocular surface squamous neoplasia employing the biopore membrane. Br J Ophthalmol 2001;85:154-8.  Back to cited text no. 9


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]

  [Table 1], [Table 2], [Table 3]


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