|Year : 2013 | Volume
| Issue : 2 | Page : 87-90
Impression cytology to study conjunctival epithelial morphology after pterygium excision with mitomycin-c and conjunctival autograft, limbal autograft (A Pilot study)
Kavita R Bhatnagar1, MN Karandikar2, Deepa Mujumdar3, Varsha Kulkarni3
1 Department of Ophthalmology, Padmshree Dr. D Y Patil Medical College, Pimpri, Pune, Maharashtra, India
2 Department of Pathology, Bharati Vidyapeeth Medical College, Pune, Maharashtra, India
3 Department of Ophthalmology, Bharati Vidyapeeth Medical College, Pune, Maharashtra, India
|Date of Submission||06-Jan-2013|
|Date of Acceptance||25-Feb-2013|
|Date of Web Publication||20-May-2013|
Kavita R Bhatnagar
B 4/21, Brahma Aangan, Off Salunke Road, Kondhwa, Pune-411048
Source of Support: None, Conflict of Interest: None
Purpose: To compare the conjunctival epithelial morphology after two types of pterygium excision procedures. Materials and Methods: Twenty-seven eyes of 22 patients with primary pterygium were randomly assigned to bare-sclera with intra-operative mitomycin C (MMC 0.02% for 60 seconds; group I) and pterygium excision with conjunctival autografting (group II). Controls were healthy eyes of age- and sex-matched subjects. Impression cytology was performed preoperatively, and at 2 weeks, 1, 3, 6, and 12 months after surgery. At each visit, any recurrence or complications were looked for. Result: There was no recurrence with conjunctiva lautograft. Though one eye had recurrence in MMC group, none showed any serious complications like corneal or scleral melting, glaucoma, or cataract. Pterygium excision wounds healed in a similar four-stage process in both the groups, but at different rates and with different final results. The nucleus-to-cytoplasm (N/C) ratio was highest at about 1 month postoperatively in group 1 and at 2 weeks in group 2, before gradually returning to control levels. Preoperatively, the goblet cell density (GCD) in treated eyes was almost twice than that in control eyes (P = 0.001), but fell to zero immediately postoperatively. Goblet cells first appeared (with rapidly increased density) in group II. At 12 months, the mean GCD in group II was not significantly different from those in controls, whereas the mean GCD in group I was still less than that of control (p = 0.02). Conclusion: A single application of mitomycin c after pterygium excision in the concentration of 0.02% for duration of 60 seconds is not associated with serious complications. Conjunctival autograft prevents corneal recurrence more effectively than MMC. Goblet cell density is markedly reduced by mitomycin c. Even 1 year after surgery, the ocular surface remains abnormal with respect to epithelial phenotypes in eyes treated by any of the two techniques.
Keywords: Conjunctival epithelial morphology, impression cytology, pterygium excision
|How to cite this article:|
Bhatnagar KR, Karandikar M N, Mujumdar D, Kulkarni V. Impression cytology to study conjunctival epithelial morphology after pterygium excision with mitomycin-c and conjunctival autograft, limbal autograft (A Pilot study). J Clin Ophthalmol Res 2013;1:87-90
|How to cite this URL:|
Bhatnagar KR, Karandikar M N, Mujumdar D, Kulkarni V. Impression cytology to study conjunctival epithelial morphology after pterygium excision with mitomycin-c and conjunctival autograft, limbal autograft (A Pilot study). J Clin Ophthalmol Res [serial online] 2013 [cited 2019 Oct 22];1:87-90. Available from: http://www.jcor.in/text.asp?2013/1/2/87/112176
Conjunctival impression cytology was introduced by Egbert et al. in 1977.  Impression cytology refers to the application of cellulose acetate filter material to the ocular surface to remove the superficial layers of the conjunctival epithelium.  Morphology of the conjunctival epithelium is preserved, which permits the use of a limited range of histological techniques.  The purpose of this study was to compare the process of conjunctival epithelial regeneration after two types of pterygium excision procedures that is bare sclera with intra-operative mitomycin c and pterygium excision with conjunctival auto graft.
| Materials and Methods|| |
This pilot was a prospective, randomized, comparative case series study conducted between October 2009 and June 2011 in a tertiary care teaching hospital where 27 eyes of 22 patients with primary progressive pterygium with no variation in size of pterygium and extent of corneal involvement were studied. These eyes were randomly assigned to group I, bare sclera with intra-operative mitomycin-c 0.02% for 60 seconds (13 eyes) and group II, pterygium excision with conjunctival autograft taken from superior peri-limbal area of same eye (14 eyes). Group III (Controls) were healthy eyes of age- and sex-matched subjects (total 27 eyes).
Patients with other ocular surface disorders were excluded from the study. A written informed consent was taken from the study subjects after explaining the procedure and follow-up protocol. Institutional ethics committee approval was taken before starting the study.
Patients underwent pterygium excision by bare sclera technique in the operation theater under surgical microscope. After preparing and draping the eye in normal sterile fashion, the lids were separated using eye speculum. Surface anesthesia was achieved with proparacaine 1% eye drops. Lignocaine solution 0.5 ml of 2% was injected into the pterygium. The head of the pterygium was grasped with toothed forceps, and excision was started about 0.5 mm ahead of the pterygium using No.15 Bard-Parker blade and carried down clearly to limbus till corneal surface was smooth and regular. The conjunctival and sub-conjunctival tissue was then cleaned over the sclera towards the insertion of the medial rectus muscle taking care not to injure it. Excision of pterygium was carried out upto 4mm posterior to the limbus. Hemostasis was achieved with wet field bipolar cautery.
In the MMC group (group I), intra-operative application of mitomycin c was done using 5 mm surgical sponge soaked with 0.02% mitomycin c solution and placed on the exposed scleral surface and in the fornix recesses beneath the cut margins of the conjunctiva for 60 seconds. After the sponge was removed, the eye surface was irrigated with 200 ml of balanced salt solution.
In the conjunctivalautograft group (group II), graft size was measured to cover entire bare sclera with Castroviejo caliper, and marking was done using trypan blue dye. Optimum size of the limbalconjunctival graft was taken from superior part of the same eye and was spread on to the cornea to help ensure correct tissue orientation and was then transferred on to the bare sclera without lifting the tissue even once. The graft was secured with 10-0 nylon sutures.
Post-operative treatment included steroid and antibiotic combination eye drops four times a day and ointment at bed time. Patients were followed up at day 1, week 1 and 2, and 1, 3, 6, and 12 months post-operatively. At each visit, complete ophthalmological examination was carried out with special attention to recurrence and complications of mitomycin c such as corneal edema, glaucoma, corneal or sclera melting, superficial punctuate keratitis, and cataract.
Recurrence was defined as a fibro-vascular growth beyond the limbus into the cornea upto 1 mm.
Conjunctival impression cytology was done pre-operatively and post-operatively at 2 weeks, 1, 3, 6, and 12 months. Conjunctival impression cytology was performed using millipore filter paper (0.025-0.22 microns) of 3 × 3 mm pieces. Filter paper was removed with a peeling motion and then applied to a clean glass slide at room temperature. The impression was transferred onto the glass slide by uniform gentle pressure. Fixing was done using 95% ethanol and 1% formalin. Specimens were subjected to periodic-acid-schiff (PAS) andpapanicolaou (PAP) stains. Samples were studied under low and high power of compound microscope for epithelial cell morphology, nuclear/cytoplasmic (N/C) ratio, and goblet cell density (GCD).
Results were graded according to Nelson grading.  Statistical analysis was carried out with the Chi-square test.
| Results|| |
All patients had successful pterygium removal with no corneal recurrence in limbal conjunctival auto graft group while one out of 14 patients showed recurrence in mitomycin c-treated group. Mild side-effects such as pain and photophobia were observed in mitomycin c group in first 1-2 weeks. Corneal changes in the form of superficial punctuate keratitis was seen in one eye in mitomycin c group, which subsided within 2 weeks post-operatively. Wound healing was delayed in most of the cases in mitomycin c group. None of the eyes showed any serious complications like corneal or scleral melting, glaucoma, or cataract.
Pterygium excision wounds healed in a similar four-stage process in both the groups, but at different rates and with different final results. The nucleus-to-cytoplasm N/C ratio was highest at about 1 month post-operatively in group I, and at 2 weeks in group II, before gradually returning to control levels [Table 1]. Pre-operatively, the goblet cell density GCD in treated eyes was almost twice than that in control eyes (p 0.001) but fell to zero immediately post-operatively. Goblet cells first appeared (with the most rapidly increased density) in group II, followed by group I (which was four-fold less than pre-operative GCD). At 12 months, the mean GCD in group II was not significantly different from those in controls, whereas the mean GCD in group I was still less than that of control (P 0.02) [Table 2].
Impression cytology study under compound microscope revealed following findings. Controls showednormal conjunctival epithelium: Nelson grade-o with numerous plump ovaloid goblet cells (arrow) with PAS +ve cytoplasm with periodic acid schiff stain in 22 (%) eyes [Figure 1]. Group II showed highest N/C ratio with dysplastic epithelial cells with karyomegaly at two weeks [Figure 2]. Group I showed highest N/C ratio with dysplastic epithelial cells with karyomegaly at one month post-operatively [Figure 3]. Group II showed numerous goblet cells with strongly PAS-positive cytoplasm at 12 months post-operatively, but epithelial cell morphology still showed few dysplastic cells [Figure 4]. Group I showed non-goblet conjunctiva with dysplastic cells at 12 months post-operatively [Figure 5].
|Figure 1: Low power of normal conjunctival epithelium: Nelson gr-o with numerous plump ovaloid goblet cells (arrow) with PAS +ve cytoplasm, periodic acid Schiff stain|
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|Figure 2: Group II-pterygium excision with counjuctivalautograft: Dysplastic epithelial cells with karyomegaly (arrow), some with oval nucleus, nucleoli seen, pap stain, 400X. (two weeks post-operative)|
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|Figure 3: Group I-pterygium excision with mytomycin-c: Dysplastic epith cell with karyomegaly, with 2 PAS +ve goblet cells (arrow), oil immersion, 1000X, PAS stain. (one month post-operative)|
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|Figure 4: (Group II pterygium excision with counjuctivalautograft), conjunctival squamous epithelium with few dysplastic cells, numerous goblet cells (arrow) with strongly PAS+ve cytoplasm, 400X; PAS stain. (12 months post-operative)|
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|Figure 5: (Group I-pterygium excision with mytomycin-c), non-goblet conjunctiva squamous epithelium with few dysplastic cells (arrow), 400X; PAS stain (12 months post-operative)|
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| Discussion|| |
Pterygium is a worldwide condition commonly seen in the Cameron pterygium belt located between 37 degree north and south of equator.  Pterygium is a condition whose origin, development, and tendency to recur, all offer problems to ophthalmic surgeons. The recurrent pterygium is more aggressive in nature; therefore, it is also called as sleeping tiger. Surgical excision is the treatment of choice.
Simple excision of the pterygium alone has a very high rate of recurrence that is about 30-70%.  Various adjunctive strategies such as irradiation treatment, anti-metabolites, conjunctivalautograft, limbalautograft, and amniotic membrane graft have been employed over the years to reduce the high recurrence rates with mixed success. ,
The use of intra-operative application of mitomycin c gives a high success rate; however, serious complications have been reported. ,
Kunimoto and Mori first described the use of mitomycin c eye drops in Japan in 1963. In an attempt to reduce the toxicity associated with mitomycin c eye drops use, possibly related to patient overdosing, several surgeons now advocate a single intra-operative application of mitomycin c. Most studies have advocated 0.04% to 0.02% doses and duration of 3-5 minutes. Cheng HC et al. demonstrated efficacy and safety of intra-operative 0.02% mitomycin c for lesser duration of 30 seconds, with a recurrence rate of 7.95 with mean follow-up of 27.3 + 4.1months. 
We studied the process of conjunctival epithelial regeneration after two types of pterygium excision procedures that is single intra-operative application of mitomycin c and conjunctival autograft with the help of conjunctival impression cytology.Impression cytology provides a flat mount of an area as large as the size of the applied filter paper with well-preserved morphology. Conjunctival smears destroy much of the morphological information. , Conjunctival biopsies provide information on a relatively small sample of the surface epithelium. ,
Conjunctival impression cytology is based purely on squamous epithelial and goblet cell abnormalities of exfoliated cells from conjunctival epithelium obtained by impression on millipore filter paper and stained with PAP and PAS stain. The changes in epithelial cell morphology, nuclear/cytoplasmic (N/C) ratio, and goblet cell density can be graded according to the scheme described by Nelson et al. According to this scheme, grade 0 is (normal), in which epithelial cells are small and round, nuclei large with N/C ratio 1/2, goblet cells numerous, plump ovaloid with intensely PAS +vecytoplasm; Grade I is (slightly abnormal), in which epithelial cells are slightly longer and polygonal, nuclei small with N/C ratio 1/3, goblet cells lower in nos. PAS +vecytoplasm; Grade II is (abnormal), in which epithelial cells are larger and polygonal, N/C ratio 1/4 - 1/5, goblet cells significantly lower in nos., less intensely PAS +vecytoplasm.; Grade III is (significantly abnormal), in which epithelial cells are larger and polygonal, nuclei small andpyknotic, N/C ratio 1/6 with goblet cells absent. 
The nucleus-to-cytoplasm (N/C) ratio was first raised in group II (conjunctival auto graft). Goblet cells first appeared (with the most rapidly increased density) in group II. The mean GCD in group II was not significantly different from those in controls, whereas the mean GCD in group I (MMC) was still less than that of control. The surface epithelium morphology remained abnormal even at the end of 12 months post-operatively. Our findings corroborate with findings of Tseng HC et al.  Solomon J also studied effect of Mitomycin-c in pterygium surgery and found a marked decrease in goblet cell density but no abnormality in conjunctival cell morphology on long term follow-up. 
Limitations of this study: Since this was a pilot study carried out on 27 eyes (13 and 14 for each group respectively), more number of samples needs to be studied to corroborate our findings.
| Conclusion|| |
A single application of mitomycin c after pterygium excision in the concentration of 0.02% for a duration of 60 seconds is not associated with serious complications. Conjunctival autograft prevents corneal recurrence more effectively than MMC. Impression cytology revealed marked decrease in goblet cell density in mitomycin c-treated cases while goblet cells returned to normal size, shape, and density so also the staining characteristics in conjunctivalautograft group. Even 1 year after surgery, the ocular surface remained abnormal with respect to epithelial phenotypes in eyes treated by any of the two techniques.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]