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G. P. Williams, A. K. O. Denniston, K. S. Oswal, P. J. Tomlins, R. J. Barry, S. Rauz, S. J. Curnow; Age Related Changes in Leukocyte Populations in the Healthy Human Conjunctival Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2363.
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The conjunctiva is a highly specialised mucosal surface that, like other mucosa, contains a number of different leukocyte populations. These have been identified mainly by immunohistochemical studies of conjunctival biopsy material, preventing both detailed and longitudinal analysis. Our aim was to utilise impression cytology (IC) with multi-colour flow cytometry to characterise in detail the frequency of leukocyte subsets in the healthy human conjunctival epithelium and how these change with age.
This study was conducted following ethical approval and in accordance with the declaration of Helsinki. Conjunctival cells were collected using IC with two sterile Supor filters per eye, following instillation of 0.4% Oxybuprocaine from 60 eyes of 30 healthy volunteers (median age 61 years [range 21-83]). Cells were recovered by gentle agitation. Matched peripheral blood was collected in EDTA prior to red cell lysis. Cells were stained with fluorochrome-conjugated antibodies in two 9-colour panels to describe T cells and other leukocytes before flow cytometric analysis.
Lymphocytes comprised 71% [38-86] of the total CD45+ leukocyte population of the conjunctiva, with 69% of these CD3+CD56- T cells. Furthermore, and in contrast to peripheral blood, the dominant population was TCRαβ+ CD8αβ+ (80%) with only 10% CD4+ cells. In the conjunctiva, with age, there was a significant increase in the proportion of NK cells (r=0.41; p=0.02) and a decrease in the proportion of T cells (r=-0.45; p=0.01), although there was an increase in the total number of CD4+ cells (r=0.6; p<0.001). This resulted in an increase in the CD4:CD8 ratio (r=0.5;p=0.01) in the ocular surface with age, which was also observed in peripheral blood.
Analysis of leukocytes of the conjunctival epithelium by IC and multi-colour flow cytometry revealed that the dominant population was TCRαβ+ CD8αβ+ T cells. The distributions however changed with age in both the blood and conjunctiva, in particular with an alteration of CD4+, CD8αβ+ and NK cell populations. It is not clear whether this reflects changes in the blood or a change in the environment with age. This technique offers a non-invasive procedure to determine detailed cellular characteristics of conjunctival epithelium in health, but more importantly, it offers potential for longitudinal sampling in patients with ocular surface disease.
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