Journal of Cytology
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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 25  |  Issue : 2  |  Page : 50-54
Conjunctival cytology in glaucomatous patients using long-term topical therapy

1 Department of Pathology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh, India
2 Department of Ophthalmology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh, India

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Background: Long-term use of antiglaucoma drugs induces adverse changes on the conjunctival surface.
Aim: To evaluate the cytological changes in the conjunctival scrape smears of patients receiving long-term antiglaucoma medication and their histopathological correlation.
Materials and Methods: Conjunctival scrape smears were taken from the eyes of patients on long-term antiglaucoma therapy for over three months (n = 75), patients taking antiglaucoma medication for less than three months (n = 100) and from glaucomatous patients in whom trabeculectomy was done as a primary procedure. Inflammatory cell counts, fibroblasts, and the degree of metaplasia were then evaluated both cytologically and histologically. The t-test was used to determine the predictive values of these parameters for the surgical outcome of trabeculectomies.
Results: Long-term use of antiglaucoma therapy leads to a higher stage of metaplasia with an increase in the number of fibroblasts, subepithelial collagen deposition, and inflammatory infiltrate within the substantia propria of the conjunctiva.
Conclusions: Long-term antiglaucoma medications induce a significant degree of metaplasia in the conjunctival surface that adversely affects the outcome of filtration surgery.

Keywords: Conjunctiva; glaucoma; metaplasia; surgery.

How to cite this article:
Sherwani RK, Khan R, Pervez MA, Tauheed A, Ashraf H, Jilani F A. Conjunctival cytology in glaucomatous patients using long-term topical therapy. J Cytol 2008;25:50-4

How to cite this URL:
Sherwani RK, Khan R, Pervez MA, Tauheed A, Ashraf H, Jilani F A. Conjunctival cytology in glaucomatous patients using long-term topical therapy. J Cytol [serial online] 2008 [cited 2022 Dec 1];25:50-4. Available from:

   Introduction Top

Glaucoma is one of the leading causes of blindness and can have varied clinical and histopathological presentations. With advances in health care, it has been observed that prolonged and indiscriminate use of drugs may have many deleterious effects on tissues. The same is true for antiglaucoma drugs with which the initial response may be good in terms of intraocular pressure control, but sooner or later, these drugs fail and a laser or surgical intervention is required.

There is growing evidence that long-term treatment by antiglaucoma drugs may adversely affect the outcome of filtration surgery. [1],[2] Studies conducted in chronic users of topical antiglaucoma drugs have revealed increases in the number of fibroblasts and inflammatory cells in the conjunctival substantia propria, decreases in the number of epithelial goblet cells, and the induction of a degree of epithelial metaplasia with ultrastructural changes. [3] All these changes promote fibrosis of filtration blebs with disruptions in postoperative wound healing. [4]

In the present study, we have examined the cytological and histological effects of long-term treatment with antiglaucoma drugs on the cell content of conjunctiva. The conjunctival changes were correlated with the results of filtration surgery in cases with or without prolonged topical medication.

   Materials and Methods Top

Two hundred eyes of 150 glaucoma patients selected from the glaucoma clinics from our Institute of ophthalmology, were divided into three groups:

Group I: Glaucomatous patients using topical antiglaucoma drugs for < three months (nonoperative group), comprising of 100 eyes.

Group II: Glaucomatous patients who were on topical antiglaucoma therapy for long periods ( >three months) and in whom trabeculectomy was subsequently performed due to the failure of drugs to control intraocular pressure (IOP) (operative group), comprising of 75 eyes.

Group III: Glaucomatous patients in whom trabeculectomy was done as a primary procedure (operative group), comprising of 25 eyes.

After taking detailed clinical history and thorough examination of the patients, a scrape smear of the bulbar conjunctiva was made by a sterilized scraper after anesthetizing it with one drop of 4% xylocaine on an albuminized slide in one drop of 85% normal saline. The slides were then air-dried, fixed in 95% ethanol, and stained with modified Papanicolaou's stain.

The prepared smears were then examined for loss in the goblet cell population, changes in the nuclei and cytoplasm of epithelial cells, and the degree of keratinization. These changes were then graded in six different cytological stages according to Tseng's classification. [5]

Cytological grading (Tseng's classification) [5]

Stage 0: Normal conjunctival epithelium, moderate number of goblet cells; nongoblet epithelial cells have blue-green cytoplasm, N:C ratio 1:1, goblet cell density (GCD) variable.

Stage I: Early loss of goblet cells, no keratinization, mild enlargement of nongoblet epithelial cells, N:C ratio 1:2 to 1:3, GCD decreased.

Stage II: Total loss of goblet cells, no keratinization, nongoblet epithelial cells show squamoid changes, N:C ratio 1:4, GCD markedly decreased or absent.

Stage III: Early and mild keratinization; squamoid changes of all epithelial cells with metachromatic change in cytoplasm, pinkish color, N:C ratio 1:6.

Stage IV: Moderate keratinization, squamoid changes as in stage III; more cells with densely packed keratin; kerato-hyaline granules and pyknotic nuclei, N:C ratio 1:8.

Stage V: Advanced keratinization; increased keratinization with shrunken cytoplasm and densely packed keratin filaments; markedly pyknotic or enucleated squamous cells and keratin debris.

GCD described in the classification given above is not applicable to the present study because the samples reveal exfoliative cytology wherein the spatial arrangement of cells has been lost.

A conjunctival biopsy was then taken from the infrabulbar region, 3-5 mm away from the limbus before peribulbar anesthesia was given for trabeculectomy. The slides were stained with hematoxylin and eosin (H and E) and examined under a light microscope for cell morphology, squamous metaplasia, the number of goblet cells, degree of keratinization, number of fibroblasts, macrophages, lymphocytes, plasma and mast cells, and granulocytes in the conjunctival lamina propria as well as for the presence of fibrosis and inflammatory edema. Cells were counted in 40 magnification. Inflammatory cell counts were performed in the conjunctival stroma in four different fields and averaged to obtain the representative mean cell counts. Inflammatory cells in blood vessels were not counted. Van Gieson's stain for collagen was done wherever possible. Squamous metaplasia was graded histologically in correlation to the cytological grading.

In our study, we have determined, both cytologically as well as histologically, the inflammatory cell counts along with fibroblasts and the degree of metaplasia in patients on antiglaucoma therapy. The t -test was used to determine predictive values of these parameters for the surgical outcome of trabeculectomies.

   Results Top

Two hundred eyes of 150 patients were divided into three study groups (as elaborated in Materials and Methods). Females were found to be more commonly affected in all three groups with the maximum number of cases belonging to the age group of 46–55 years.

Intergroup comparisons of the observed grade of metaplasia showed comparable results between groups I and III ( i.e., nondrug users) with the maximum number of cases between stages I and III [Figure 1]. Cases in group II (chronic drug users) however, showed metaplasia of grades IV and V [Table 1],[Figure 2].

[Table 2] shows intergroup variation between various cell counts and inflammatory edema. Statistically significant ( P < 0.001) increases in fibroblasts were found in group II (37.5 12.5) [Figure 3] compared to groups I and III (21.01 14.04 and 21.5 4.5), respectively. Mean lymphocyte and plasma cell counts were also found to be significantly raised in group II as compared to groups I and III ( P < 0.001). Mild inflammatory edema was found in 78% of group I cases with 17% showing no edema. Almost similar results were seen in group III with 70% of the cases having mild edema, 2% having moderate while 28% having no edema. However, 53.3% of group III cases showed moderate to severe edema, with none having no edema. A subepithelial collagen deposit was seen in 73.3% of group II patients [Figure 4] as compared to 5.55 and 7.25% in groups I and III, respectively.

Histological and cytological comparison of the grade of metaplasia showed no significant differences [Table 3].

It was also found that although combination therapy revealed a lower percentage in all stages of metaplasia as compared to single drug therapy when the duration of therapy was disregarded. However, high grades of metaplasia were seen in combination therapy when compared to other groups with the same duration of therapy.

At the three to six months' follow-up visit, mean postoperative intraocular pressures over 21 mm Hg for all eyes in groups I and III were seen in 22 and 8% of eyes, and 58.6% in eyes belonging to group II. Failure rate of trabeculectomy was about 12% in cases of group II, while being minimal (2 and 4%) in cases belonging to groups I and III respectively [Table 4].

   Discussion Top

Although the use of antiglaucoma drugs for newly diagnosed cases of glaucoma has become common practice, recent studies question this traditionally held belief and have proposed early filtration surgery. [6],[7]

Many authors have reported various adverse external ocular effects of antiglaucoma drugs after their prolonged usage especially in the form of changes in the conjunctival surface epithelium and inflammatory cell counts. Thus, the success rate of filtration surgery is decreased by enhancing the risks of subconjunctival fibrosis and bleb formation. Consistent with other reports, we found loss of goblet cells and increased conjunctival epithelial metaplasia in those using chronic topical antiglucoma drugs. [8],[9]

Baun et al . [10] suggested that topical antiglaucoma treatment for four years does not induce morphological changes of inflammation or fibrosis of the conjunctival. In contrast, our study showed these changes to occur within three months. Similar findings of an increased degree of metaplasia have also been documented in various studies with prolonged antiglaucoma therapy. [3],[4],[9]

Similar to Broadway et al. [11] and Pazarowska et al. , [8] our study also showed that preoperative conjunctival biopsy from the patients whose surgery eventually failed contained more fibroblasts, macrophages, lymphocytes, and pale cells. This provided evidence of the potentially causative relationship between conjunctival changes and surgical failure. These findings are contrary to those of Gwynn et al. [12] and Arici et al ., [13] because the only statistical difference they could establish was for goblet cells which significantly decreased in the group showing more difficult postoperative IOP control.

We also found that with increasing degrees of metaplasias in chronic drugs users, postoperative IOP control was poor. All the eyes which showed failure of trabeculectomy in nondrug users had stage I metaplasias while metaplasia was beyond stage III in chronic drug users.

Our study found an increase in subepithelial collagen in 73.3% of group II cases as compared to 5.5% group I cases, a finding similar to that of Meitz et al. , [14] They also reported an increase in the thickness of subepithelial collagen following treatment with preserved drugs for three months in rabbit conjunctival. Jilani et al . [15] showed increases in the subepithelial collagen band with an increase in the duration of topical antiglucoma therapy.

Cytological and histomorphological grading of metaplasias of the conjunctival epithelium after long-term topical antiglaucoma therapy, showed comparable results.

Taking IOP >21 mm Hg as a criterion for the failure of trabeculectomy, it was found that the failure rate of trabeculectomy was about 1-2% in nondrug users whereas it was about 12% in chronic drug users. Our findings are comparable with the results of a previous study conducted by Jilani et al . [15] Miller et al . [16] also reported a lower success rate of trabeculectomy in patients who had previously received multiple antiglaucoma topical agents. Thus, the number of preoperative antiglaucoma medications used and any history of previous topical therapy were the adverse factors for the outcome of trabeculectomy. [17]

In conclusion, it was found that long-term use of antiglaucoma therapy had an adverse effect on the outcome of filtration surgery, and therefore, an early surgical intervention is preferred despite the risks associated with surgery.

   References Top

1.Baudouin C. Mechanism of failure in glaucoma filtering surgery: A consequence of antiglaucomatous drugs? Int J Clin Pharmacol Res 1996;16:29-41.  Back to cited text no. 1  [PUBMED]  
2.Stefan C, Nenciu A, Balas M, Neac?u A, Dachin L, Ardelean C. Conjunctival changes related to topical treatment and effectiveness of surgery in glaucoma. Oftalmologia 2003;59:19-23.  Back to cited text no. 2    
3.Hong S, Lee CS, Seo KY, Seong GJ, Hong YJ. Effects of topical antiglaucoma application on conjunctival impression cytology specimens. Am J Ophthalmol 2006;142:185-6.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Nenciu A, Stefan C, Ardelean C. Structural and immunohistochemical changes of conjunctiva induced by topical glaucoma medication. Oftalmologia 2004;48:35-42.  Back to cited text no. 4  [PUBMED]  
5.Tseng SC. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology 1985;92:728-33.  Back to cited text no. 5  [PUBMED]  
6.Liesegang TJ. Conjunctival changes associated with glaucoma therapy: Implications for the external disease consultant and the treatment of glaucoma. Cornea 1998;17:574-83.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Anand A, Negi S, Khokhar S, Kumar H, Gupta SK, Murthy GV, et al . Role of early trabeculectomy in primary open-angle glaucoma in the developing world. Eye 2007;21:40-5.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Pozarowska D, Taczolowsk J, Wozmak F. The evaluation of morphological status of bulbar conjunctiva after long term antiglucoma drug therapy. Klin Oczna 1999;101:455-8.  Back to cited text no. 8    
9.Nuzzi R, Finazzo C, Cerruti A. Adverse effects of topical antiglaucomatous medications on the conjunctiva and the lachrymal response. Int Ophthalmol 1998;22:31-5.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Baun O, Heegard S, Kessing SV, Prause JU. The morphology of conjunctiva after long term topical antiglucoma treatment: A quantitative analysis. Acta Ophthalmol Scand 1995;73:242-5.  Back to cited text no. 10    
11.Broadway DC, Grierson J, O'Brien C, Hitchings RA. Adverse effect of topical antiglucoma medication I: The conjunctival cell profile. Arch Ophthalmol 1994;112:1437-45.  Back to cited text no. 11    
12.Gwynn DR, Stewart WC, Pitts RA, McMillan TA, Hennis HL. Conjunctival structure and cell counts and the results of filtering surgery. Am J Ophthalmol 1993;116:464-8.  Back to cited text no. 12  [PUBMED]  
13.Arici MK, Demircan S, Topalkara A, Gulei C, Aker H, Arici DS. Effect of conjunctival structure and inflammatory cell counts on intraocular pressure after trabeculectomy. Ophthalmologica 1999;213:371-5.  Back to cited text no. 13    
14.Meitz H, Niesen U, Krieglstein GK. The effect of preservatives and antiglucoma medication on histopathology of conjunctiva. Graefes Arch Clin Exp Ophthalmol 1994;232:561-5.  Back to cited text no. 14    
15.Jilani FA, Ashraf H, Sherwani RK, Kulshrestha V. Reversal of adverse effect of topical antiglucoma therapy on the conjunctiva. Asian J Ophthalmol 2001;1:11-4.  Back to cited text no. 15    
16.Miller MJ, Rice NS. Trabeculectomy combined with beta irradiation for congenital glaucoma. Br J Ophthalmol 1990;75:748-90.  Back to cited text no. 16    
17.Longstaff S, Wormald RP, Mazover A, Hitchings RA. Glaucoma triple procedures: Efficacy of intraocular pressure control and visual outcome. Ophthalmic Surg 1990;21:786-93.  Back to cited text no. 17  [PUBMED]  

Correspondence Address:
Roobina Khan
Hafeez Manzil, Marris Road, Aligarh - 202 001, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9371.42442

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4]

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