Sixteen consecutive patients with VKC (mean age, 11.2 ± 4.9 years; range, 7–22) and 10 normal subjects were included in the study. Patients with VKC (12 male and 4 female) were in an active disease phase and had been free of treatment for at least 3 days. The control group was age matched (mean age, 16.5 ± 5; range, 10–24) and included six males and four females. No subject in the control group used contact lenses or had any inflammatory signs and symptoms. The research adhered to the tenets of the Declaration of Helsinki. A written informed consent was obtained from all subjects or their parents before tears were collected. Diagnosis of VKC was based on clinical history and evaluation of signs and symptoms. Patients with VKC were tested for skin test reactivity and the presence of specific IgE in serum for common environmental allergens (CAP-System; Pharmacia & Upjohn, Uppsala, Sweden). A subjective clinical score (0–10) was given to each patient by one of the investigators (AL), to evaluate the overall severity of the disease. Clinical scores (0–4) for each ocular symptom (itching, tearing, photophobia, and foreign body sensation) and each sign (conjunctival erythema, mucous discharge, papillae, limbal infiltrates, and corneal epithelial disease) were assigned at the time of the visit. In particular, corneal disease was scored as follows: 1+, fine superficial epithelial defects; 2+, confluent epithelial defects; 3+, oval corneal ulcers; and 4+, oval corneal ulcers with plaque. Papillae were scored as follows: 1+, mild hyperemic scattered papillae; 2+, moderate hyperemic swollen papillae 1 mm size; 3+, at least two to three giant papillae more than 1 mm size; and 4+, giant papillae covering the superior tarsal plate.
Tear samples were collected from the outer canthus with a microcapillary tube and centrifuged for 10 minutes at 1000 rpm to separate the cells from the tear fluid. Tear volume ranged from 30 to 200 μL. Supernatants were stored at −80°C until assaying. Cell pellets were resuspended in 20 μL aliquots, and the percentage of eosinophils, neutrophils, and lymphocytes present in five microscopic fields of 0.15 mm2 were counted on precolored slides (Testsimplet; Roche Molecular Biochemicals, Mannheim, Germany) using a microscope at high-power (400×) magnification (Carl Zeiss Meditech, Oberkochen, Germany).
In two of these young patients (AF, UT), the corneal plaque was surgically removed under general anesthesia. A written informed consent was obtained from the parents. A superficial keratectomy was performed, scraping off the plaque with a knife. Plaque debris were snap frozen with OCT in liquid nitrogen and maintained at −70°C for additional immunohistochemistry (IHC).