CLINICAL SCIENCE

Vernal Keratoconjunctivitis With Limbal Stem Cell Deciency

Virender S. Sangwan, MD,* Vandana Jain, MD,* Geeta K. Vemuganti, MD, and Somasheila I. Murthy, MD*
individuals. The chronic nature and prolonged duration of the disease may be responsible for this secondary ocular surface affection, leading to the clinical manifestation of LSCD. Key Words: limbal stem cell deciency, vernal keratoconjunctivitis, squamous metaplasia, amniotic membrane graft, limbal stem cell transplantation (Cornea 2011;30:491496)

Purpose: To describe the clinical features of patients affected with

vernal keratoconjunctivitis (VKC) with associated limbal stem cell deciency (LSCD).

Methods: In a retrospective observational case series, data relating

to 49 eyes of 27 patients with VKC and features of LSCD seen from January 2000 to August 2005 at the Cornea Service of LV Prasad Eye Institute were reviewed. Detailed history, visual acuity at presentation, clinical ndings, disease activity, duration of complaints, and extent of LSCD and management were noted. The presence of concurrent keratoconus and other ocular or systemic disorders if any were recorded.

Results: During this period, 2225 patients with VKC were seen at our institute. Of the 49 eyes, (1.2%) showed features of LSCD. The mean age was 21.37 6 10.14 years. The male to female ratio was 4 to 1. Twenty-two cases had bilateral disease, and 5 had unilateral affection. The visual acuity at presentation ranged from hand motions to 20/20. The duration of disease ranged from 1 to 25 years, with a mean of 10 6 6.66 years. Total LSCD was seen in 33 eyes (67.3%), and partial LSCD was seen in 16 (33%). Disease at the time of presentation was active in 15 cases (55.5%) and was quiescent in 12 cases (44.5%). Pannus resection with amniotic membrane graft was done in 5 eyes. Live-related allolimbal transplantation was done in 6 eyes of 3 patients. VKC patients with or without associated LSCD had similar characteristics, but the age of presentation was lower in patients without LSCD (P = 0.0005, unpaired Student t test), and the duration of disease was shorter (P , 0.0001, unpaired Student t test). Conclusions: LSCD seems to be one of the complications of longstanding VKC, contributing to severe visual impairment in young
Received for publication October 23, 2008; revision received June 24, 2009; accepted October 13, 2009. From the *Cornea and Anterior Segment Service, LV Prasad Eye Institute, Hyderabad, Andhra Pradesh, India; Cornea and Anterior Segment, Aditya Jyot Eye Hospital, Mumbai, Andhra Pradesh, India; and Ophthalmic Pathology Service, LV Prasad Eye Institute, Hyderabad, Andhra Pradesh, India. Supported by Hyderabad Eye Research Foundation, Hyderabad, Andhra Pradesh, India. Presented at the American Academy of Ophthalmology Annual Meeting, Denver, Colorado November, 2006. The authors have no nancial interest in any of the issues or products referred to in this article. There is no conict of interest. Reprints: Virender S. Sangwan, LV Prasad Eye Institute, LV Prasad Marg, Banjara Hills, Hyderabad 500 034, Andhra Pradesh, India (e-mail: vsangwan@lvpei.org). Copyright 2011 by Lippincott Williams & Wilkins

ernal keratoconjunctivitis (VKC) is a chronic inammatory disease of the ocular surface, affecting young children.1 Two dramatic aspects of this disease are the morphological changes in the tarsal conjunctiva accompanied by the formation of giant papillae and the limbal conjunctival changes. The rst is probably related to broblast activation and collagen hyperproduction, whereas the limbal inltrates are the result of inammatory cell accumulation and peripheral corneal brovascular pannus.2 A spectrum of signs characterizes and differentiates the 2 major clinical forms of VKC, ie, either predominant palpebral or limbal. In temperate regions, the palpebral form is the most frequent. It is associated with a personal history of atopic disease and is more common in males. It is characterized by giant papillary hypertrophy of the conjunctiva over the upper tarsal plate. Corneal epithelial macroerosions and vernal plaque are common complications that are thought to be secondary to the conjunctival disease. Limbal VKC is common in tropical and subtropical countries. It is less often associated with a personal clinical history of asthma or eczema than in temperate countries.3,4 Usually, VKC subsides with the onset of puberty, but it may occasionally become chronic and lead to permanent changes to the ocular surface with associated visual impairment.1 Earlier, We had reported 2 cases affected with VKC with limbal stem cell deciency (LSCD)5 that had beneted by limbal stem cell transplantation (LSCT). In this paper we present the prevalence and clinical spectrum of patients with this unusual but devastating complication of VKC.

METHODS
We retrospectively reviewed the records of all cases affected with VKC with associated ocular surface affection in the form of LSCD seen from January 2000 to August 2005 at the Cornea and Anterior Segment Service, LV Prasad Eye Institute, Hyderabad, a tertiary eye care center. Both active and
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quiescent forms of VKC were dened at the beginning of the study. A diagnosis of active VKC was made according to established clinical criteria based on the presence of limbal or palpebral papillae and a history of ocular itch.1 The quiescent form was diagnosed on the basis of inactive conjunctival papillae, upper tarsal conjunctival scarring, and a previous history of itching, along with the observations comunicated by the referring ophthalmologist. LSCD was diagnosed on the basis of clinical ndings of dull irregular reex of the corneal epithelium, corneal conjunctivalization, and loss of palisades of Vogt6 (Figs. 1A, B) and was conrmed by impression cytology. Corneal impression cytology was obtained by applying nitrocellulose lter paper strips onto the perilimbal area to include both the conjunctival and the corneal surfaces in 4 different quadrants. After instilling topical anesthetic eyedrops, the membrane was rmly pressed against the area to be sampled. This takes on an average of 1020 seconds. Disks were immediately placed in xative (70% ethyl alcohol, 37% formaldehyde, and glacial acetic acid in a 20:1:1 volume ratio). In the laboratory, the specimens were stained with periodic acidSchiff and hematoxylineosin stains. All specimens were viewed under light microscope for microscopy to check for the presence of goblet cells, based on which LSCD was conrmed. Total LSCD was dened as 360-degree involvement of limbal stem cells (all 4 quadrants) with clinical and

histopathological signs of stem cell deciency. Partial LSCD was dened as involvement of less than 360 degrees. In addition to the patients demographic data, a detailed history was noted, including age at the onset of the disease, seasonal variation and duration of the condition, history of allergic diseases, and occurrence of previous or concomitant ocular or systemic diseases. Visual acuity at presentation, the extent and degree of conjunctivalization, and impression cytology ndings were also recorded. Details of any earlier surgical procedures, such as amniotic membrane transplantation and/or allo-LSCT were documented. For comparison, demographic and other relevant clinical features were studied in patients with VKC seen during the study period (Table 1). The comparison group comprised clinically diagnosed cases affected with VKC without any clinical evidence of LSCD. Impression cytology was not performed in cases where there was no clinical evidence of LSCD. SPSS (Statistical Package for the Social Sciences) 8.0 for Windows (SPSS, Inc, Chicago, IL) software was used for statistical evaluation wherever required. The signicance level was set at 0.05 for all the statistical analyses.

RESULTS
From January 2000 to August 2005, a total of 2225 patients were diagnosed with VKC using the criteria mentioned above. Of these, 27 cases (1.2%) affected with VKC with partial or total LSCD were identied (Table 2). The age ranged from 9 to 48 years, with a mean of 21.37 6 10.14 years. The series included 22 men and 5 women with a male to female patient ratio of 4 to 1, indicating a male predominance. Twenty-two cases had bilateral disease, and 5, unilateral. The duration of disease ranged from 1 to 25 years, with a mean of 10 6 6.66 years. Sixteen patients (60%) reported that symptoms persisted all around the year. Disease at the time of presentation was active in 15 cases (55.5%) and quiescent in 12 (44.5%). In patients with active disease, 11 (73%) had the mixed form and 4 (27%) had predominantly limbal disease. In none of the patients was the disease predominantly palpebral. The major symptoms mentioned by most patients were itching and photophobia. Fourteen patients complained of decreased vision in one or both eyes (Table 2). VKC was the only clinical manifestation and was not associated with any other allergic diseases. Four cases (15%) had associated keratoconus. Two cases (7.5%) had steroidinduced glaucoma, and 1 case (3.7%) had associated cataract. No history of concomitant or previous systemic disease was noted.
TABLE 1. Comparison Data of VKC With and Without LSCD
VKC VKC With LSCD Male/female ratio 7:1 4:1 Mean age at presentation (yrs) 14.75 6 6.2 21.57 6 10.14 (P = 0.0005) Duration of disease at 1.93 6 2.37 10 6 6.66 (P , 0.0001) presentation (yrs) Associated atopic disease, n (%) 20 (1%) Nil Associated keratoconus, n (%) 46 (2.3) 4 (15)

FIGURE 1. A, Right eye of the patient showing 360-degree conjunctivalization with associated corneal scarring. B, Upper tarsal conjunctiva showing the presence of giant papillae (case 12).

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TABLE 2. Patients Characteristics, Treatments, and Outcomes

Visual S No Age (yrs) Sex Eye Acuity 1 17 Symptoms Disease Activity Active (limbal form) Quadrants of LSCD Systemic/Ocular Duration of Disease Disease (Yrs) Nil 5 Surgical Intervention Nil Pannus resection with AMG Nil Nil Nil

M OD 20/20 Itching and photophobia OS 20/20 M OD 20/20 Intermittent itching and OS 20/20 photophobia M OD 20/80 Itching and decreased vision M OD 20/20 Itching and photophobia OS 20/20 M OD 20/30 Itching and watering OS CFCF M OD 20/200 Itching and decreased vision OS 20/50

16

360 degree, center of cornea spared 360 degree, central cornea spared Inactive at presentation Superior quadrant and active after Superior quadrant 5 months Active (mixed form) Inferior quadrant

Nil

10

Nil

4 5 6

13 15 44

Active (mixed form) Active (mixed form) Active (mixed form)

Superior quadrant Superior quadrant Superior quadrant 360 degree 360 degree 360 degree 360 degree 360 degree

Nil Nil Steroid-induced glaucoma Steroid-induced glaucoma Dry eye Dry eye

1 3 25

36

40

26

F OD HMCF Decreased vision, Active (mixed form) OS HMCF itching, recurrent episodes of watering, and redness M OD 20/40 Intermittent Inactive episodes of redness and OS CFCF itching M OD HMCF Decreased Inactive vision and OS 20/125 itching M OD 20/40 Itching and redness F OD 20/20 Itching and photophobia OS 20/25 M OD 20/200 Itching, photophobia, OS 20/400 and decreased vision M OD 20/160 Intermittent itching and OS 20/20 decreased vision F OD 20/30 Itching and decreased vision OS 20/40 M OD 20/30 Itching, photophobia, OS 20/50 and decreased vision M OS 20/80 Itching intermittently and decreased vision M OD 20/100 Itching, redness, and watering OS 20/60 Active (limbal form) Active (mixed form)

11

Nil Nil Nil Nil Pannus resection with AMG Pannus resection with AMG Punctal occlusion Punctal occlusion

360 degree, central cornea spared 360 degree 360 degree

Nil

10

Nil Nil

10 11 12

19 48 9

Superior quadrant

Steroid-induced glaucoma Steroid-induced glaucoma Keratoconus Cataract Cataract Nil

20

Nil Nil

15 20 5

Nil Nil Nil LK + LSCT (2 times) and PK + LSCT LK + LSCT Nil Nil Nil Nil

13

21

Superior quadrant Superior quadrant Active (mixed form) 360 degree, center of cornea spared 360 degree, central cornea spared Inactive at presentation Inferior quadrant Inferior quadrant Inactive at presentation Inferior and superior quadrants Inferior and superior quadrants Inactive 360 degree 360 degree Inactive Superior quadrant

Keratoconus Keratoconus Nil Nil Nil

10

14

18

14

15

19

12

Nil Nil Nil

16

22

Nil

10

17

24

Active (mixed)

Superior and inferior quadrants Superior and inferior quadrants

Nil

14

Nil

(continued on next page)

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TABLE 2. (continued ) Patients Characteristics, Treatments, and Outcomes

Visual S No Age (yrs) Sex Eye Acuity 18 25 Symptoms Disease Activity Inactive Quadrants of LSCD 360 degree with central involvement 360 degree with central involvement 360 degree with central involvement 360 degree, central cornea spared 360 degree 360 degree Systemic/Ocular Duration of Disease Disease (Yrs) Nil Nil Nil Nil Nil Nil Keratoconus Keratoconus 10 5 10 Surgical Intervention Nil Pannus resection + AMG LSCT LSCT Nil Nil Nil Nil

F OD 20/400 Decreased vision OS 20/600

19

12

M OD HMCF Decreased vision and OS HMCF intermittent itching M OD 20/25 Itching, watering, OS 20/60 and redness M OD 20/50 Decreased vision OS CFCF

Inactive

20

18

Active (limbal)

21

28

Inactive

22

18

M OD 20/25 Itching and photophobia OS 20/25 M OD 20/400 Itching and Decreased OS 20/200 vision M OD OS F OD OS 20/25 Intermittent itching 20/25 20/30 Itching and decreased 20/40 vision M OD 20/100 Decreased OS 20/200 vision M OD 20/25 Itching and redness

23

11

24 25

19 16

360 degree with clear center 360 degree with central involvement Active (mixed form) 360 degree, center spared 360 degree, center spared Inactive (limbal form) 360 degree, center of cornea spared 360 degree, central cornea spared Inactive at 360 degree presentation 360 degree Active (mixed form) 360 degree 360 degree Burnt out Active (mixed) 360 degree 360 degree Superior quadrant

20 20

Nil

15

Nil Nil

Nil

Nil Pannus resection with AMG Nil Nil Nil Nil LSCT LSCT Nil

Nil Nil Nil Nil Nil Keratoconus

15 1

26 27

21 13

5 3

AMG, amniotic membrane graft; CFCF, counting ngers close to face; HMCF, hand movements close to face; LK, lamellar keratoplasty; PK, penetrating keratoplasty.

Stem cell deciency involved 1 to all 4 quadrants. Total stem cell deciency was seen in 33 eyes (67.3%), and partial stem cell deciency was seen in 16 eyes (33%). Impression cytology showed evidence of goblet cells on the corneal surface in the affected areas in all patients; however, 5 cases showed squamous metaplasia on both corneal and conjunctival surfaces (cases 1, 6, 8, 20, and 21). Visual acuity at presentation ranged from hand motions to 20/20. Therapies prescribed before the rst examination at our institute consisted primarily of topical mast cell stabilizers and/or corticosteroids. After presentation, depending upon the disease activity and symptomatology, treatment involved use of mast cell stabilizers, mild topical steroids, and tear substitutes. Pannus resection with amniotic membrane graft was done in 5 eyes [cases 1 (OS), 6 (OU), 18 (OS), and 23 (OS)]; however, all these eyes had a recurrence of corneal conjunctivalization (Figs. 2AD). Live-related allolimbal transplantation was done in 6 eyes of 3 patients (cases 12, 19, and 26). Of these 6 eyes, amniotic membranecultivated LSCT was performed in 5 eyes and a conjunctivo-limbal

allograft was done in 1 eye. All the 3 patients who received allolimbal transplantation were kept under systemic immunosuppression with oral cyclosporine (35 mg/kg body weight). On the last follow-up, 3 years later, 2 patients who had undergone only LSCT were seen to be maintaining the healthy ocular surface (cases 19 and 26). They showed a partial recurrence of corneal conjunctivalization in 1 quadrant, but the center of the cornea had been spared. In patient 12, who had undergone lamellar keratoplasty/penetrating keratoplasty in both eyes along with LSCT, corneal conjunctivalization recurred and the procedure had failed. Oral cyclosporine was discontinued in this patient, while the other 2 patients were maintained on oral cyclosporine at the last follow-up. The comparison group comprised patients with VKC without associated LSCD. The mean age was 14.75 6 6.2 years. There was a male preponderance. The mean duration of disease persistence was 1.93 6 2.37 years. Age of presentation was lower in these patients (P = 0.0005, unpaired Student t test), and the duration of disease was shorter (P , 0.0001, unpaired Student t test) (Table 1).
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FIGURE 2. A, Conjunctivalization of cornea seen in all quadrants with central corneal scarring, before undergoing amniotic membrane transplantation (AMT). B, Impression cytology of the inferior perilimbal area showing goblet cell on the corneal surface (below the arrow) (original magnication 310, periodic acidSchiff stain). C, Periodic acid Schiff-stained histopathological section of the excised pannus showing the presence of goblet cell on the corneal side of the pannus (below the arrow) (original magnication 310). D, Clinical photograph showing the early recurrence of conjunctivalization with corneal scarring, 5 months after amniotic membrane transplantation (case 18).

DISCUSSION
This is the rst series of patients with VKC to highlight the ocular surface affection in the form of LSCD. We had earlier reported 2 cases affected with VKC with LSCD5 who had beneted from LSCT. We now present the prevalence and clinical spectrum of a series of patients with this unusual but devastating complication. Chronic and severe VKC-induced surface damage in the form of conjunctival scarring and corneal scarring, has been reported.1 However, with increased insight into both the ocular surface and the diseases affecting it, coupled with our understanding of limbal stem cells, we have attempted to characterize 1 type of ocular surface affection and studied its clinical features in VKC patients. When pathologic insults destroy the limbal stem cells, the corneal surface invariably heals with conjunctival epithelial ingrowth, neovascularization, chronic inammation, and/or recurrent epithelial defects constituting LSCD.6 These diseases can be subdivided into 2 major categories. The rst category is characterized by direct damage to limbal stem cells as in chemical injuries. The second category includes diverse diseases such as aniridia, peripheral ulcerative keratitis, limbitis, etc., which damage the limbal stroma to such an extent that it can no longer support these cells.7 Puangsricharen and Tseng7 reported 5 cases of peripheral (limbal) inammatory disorders with cytologic evidence of LSCD. These included 3 cases with chronic limbitis and 2 with pseudopemphigoid. They reasoned that chronic limbal inammation could result in gradual loss of stem cell function because of insufcient stromal support. The pathophysiology of VKC remains unclear, although several immunological mechanisms have been proposed including immunoglobulin Emediated and lymphocyte Th2mediated responses. Eosinophils and their degranulation products also play an important role in conjunctival inammation and in the development of corneal complications such as epithelial defects and ulcers.810 In a study by Tuft et al,4
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cellular inltration of both the conjunctival stroma and the epithelium was seen in patients with VKC consistent with chronic inammation, particularly in the limbal conjunctival biopsy specimens. The long-standing nature of the condition was suggested by the presence of squamous metaplasia in some tarsal biopsy specimens, against the background of chronic inammation. In our series, patients had long-standing VKC. This chronic limbal inammation probably affected the microenvironment of the stem cells, resulting in poor stromal support. Direct damage to the stem cells by the toxic products of eosinophils and other inammatory cells in the vicinity may also have been responsible.5 In most patients, impression cytology showed evidence of goblet cells on the corneal surface suggestive of LSCD. Five cases showed squamous metaplasia of the entire ocular surface, thereby explaining the overall reduction in goblet cells. Numerous studies have shown that squamous metaplasia of ocular surface occurs in the presence of chronic inammation and irritation, often in association with dry eye, cicatrizing diseases and chronic forms of keratoconjunctivitis such as superior limbic keratitis, atopic keratoconjunctivitis, etc.1113 VKC affects young boys and tends to subside with the onset of puberty. In a subset of patients, especially in tropical countries, it may persist beyond puberty. In our study group, the mean age of patients was 21.37 6 10.14 years and 14 of 27 patients were older than 18 years of age at the initial presentation. This subset of patients with VKC, because of long-standing chronic inammation, is probably more likely to experience greater ocular surface damage. In our series, surgical intervention was deferred during the active phase of the disease. Surgical management was performed or planned in eyes with quiescent disease and visual deterioration secondary to corneal conjunctivalization and scarring. Five eyes underwent amniotic membrane graft with brovascular pannus resection. Amniotic membrane transplantation suppresses conjunctival brosis and inammation
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while promoting corneal epithelialization and differentiation.14 In our cases, pannus resection with amniotic membrane transplantation alone was found to be insufcient for ocular surface rehabilitation, and conjunctivalization recurred in all 5 eyes after initial improvement. Six eyes of 3 patients with total LSCD received live-related allo-LSCT. To reduce the incidence of immunological rejection, immunosuppression with systemic cyclosporine was prescribed.15 Patients affected with VKC with bilateral LSCD requiring allotransplantation, may develop intermittent inammation of the ocular surface, which may jeopardize the success of the procedure. Therefore, meticulous follow-up, prolonged systemic immunosuppression, and control of disease activity is required to improve the survival rate of LSCT in patients with VKC. To conclude, VKC patients do present, though rarely, with features of vision-threatening LSCD, posing a challenge to diagnosis and management. We believe that this is secondary to the chronic nature and prolonged duration of disease. As this is a retrospective study from a tertiary care hospital, the data may not be representative of the prevalence of this unusual complication. However, a prospective study could prognosticate cases affected with VKC with regards to the potential development of this vision-threatening complication. REFERENCES
1. Bonini S, Bonini S, Lambiase A, et al. Vernal keratoconjunctivitis revisited: a case series of 195 patients with long-term followup. Ophthalmology. 2000;107:11571163.

2. Loenardi A, Radice M, Plebani FM, et al. Histamine effects on conjunctival broblasts from patients with vernal conjunctivitis. Exp Eye Res. 1999;68:739746. 3. Tuft SJ, Dart JKG, Kemeny M. Limbal vernal keratoconjunctivitis: clinical characteristics and immunoglobulin E expression compared with palpebral vernal. Eye. 1989;3:420427. 4. Tuft SJ, Cree IA, Woods M, et al. Limbal vernal keratoconjunctivitis in the tropics. Ophthalmology. 1998;105:14891493. 5. Sangwan VS, Murthy S, Vemuganti G, et al. Cultivated limbal epithelial transplantation for severe ocular surface disease in vernal keratoconjunctivitis. Cornea. 2005;24:426430. 6. Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44:415425. 7. Puangsricharen V, Tseng SCG. Cytologic evidence of corneal diseases with limbal stem cell deciency. Ophthalmology. 1995;102: 14761485. 8. Bonini S, Lambiase A, Sacchetti M, et al. Cytokines in ocular allergy. Int Ophthalmol Clin. 2003;43:2732. 9. Srivastava A, Sur S, Trocme SD. The role of eosinophils in ocular allergy. Int Ophthalmol Clin. 2003;43:925. 10. Tabbara KF. Immunopathogenesis of chronic allergic conjunctivitis. Int Ophthalmol Clin. 2003;43:17. 11. Donisi PM, Rama P, Fasolo A, et al. Analysis of limbal stem cell deciency by corneal impression cytology. Cornea. 2003;22:533538. 12. Tseng SC. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology. 1985;92:728733. 13. Soong HK, Martin NF, Wagoner MD, et al. Topical retinoid therapy for squamous metaplasia of various ocular surface disorders: a multicentric, placebo controlled double masked study. Ophthalmology. 1988;95: 14421446. 14. Kim JC, Tseng SC. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995;14:472484. 15. Tsai RJ, Tseng SC. Human allograft limbal transplantation for corneal surface reconstruction. Cornea. 1994;13:389400.

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