Objective assessment of conjunctival squamous metaplasia by measures of cell and nucleus dimensions

Abstract

The main aim of this work is to present a special method for conjunctival impression cytology (CIC) and objective measures of measure cell and nucleus dimensions. CIC using Millicell® Biopore filters was obtained from 50 Caucasian adults from the nasal bulbar conjunctiva. Giemsa-stained images of cells in a monolayer were photographed with a scale bar and projected at 1,500× to generate an overlay. Measures were made of both the long (L) and short (S) dimensions of the cells and the longest dimension of the nucleus (LN). The selected images comprised sets of 10 from different individuals, which had been assigned grades 0, 1, 2, or 3 and an additional grade 0.5. L ranged from 18.4 to 65.2 μm, S from 13.1 to 47.1 μm, to give average cell shape values (L:S ratio) of between 1.259 and 1.890. L and S were smallest for grade 0 cells (group mean 19.8 ± 1.1 and 14.8 ± 1.2 μm) and largest for grade 3 cells (group 51.2 ± 7.2 and 37.2 ± 4.7 μm), with no difference in L:S ratios across the cell grades. Longest dimension of the nucleus (NL) values ranged from 8.7 to 14.5 μm, with group mean values of 9.8 ± 0.6 μm for grade 0 cells and 12.9 ± 1.2 μm for grade 3 cells. NL was nonlinearly associated with L. Measures of conjunctival cell and nucleus dimensions can be used to objectively distinguish between cells assigned different grades of squamous metaplasia, with slight nucleus enlargement being the earliest detectable change (at grade 0.5).

Contact Lens Wear and the Development of Squamous Metaplasia of the Surface Cells of the Conjunctiva

“…6). 41 These findings represent a substantial departure from the Nelson grading scale (Table 1) where it is noted that these cells have pyknotic nuclei. Using appropriate sets of cells and morphometry, it should be possible to define just how small any pyknotic nuclei might be and to assess their prevalence in CIC samples from contact lens wearers.…”

“…Although they might be considered as ÔroundÕ (or at least ÔroundishÕ), measurements reveal that these cells are mostly slightly elongated. 21,41,45,46 In Figure 5 is shown an example of grade 3 squamous metaplasia cells, at the same magnification as that in Figure 4A, to illustrate the application of an alternative approach to the objective assessment of these cells. The epithelial cells in Figure 5 are much larger than those in Figure 4A, but it is again possible to distinguish most of the cells and their nuclei so as to be able to make measurements.…”

“…41 Logically, therefore, a less invasive technique would be an in vivo technique that does not remove layers of cells onto a filter for analysis, although it does usually require the use of a topical ocular anesthetic and a coupling gel to bring the microscope objective lens into optical contact with the conjunctival surface. 50 The results from such confocal microscopy studies, to date, have however revealed that investigators have encountered similar problems to those encountered in earlier CIC studies.…”

“…Over the years, various investigators have reported their experiences that have led them to develop slightly different methods of grading the character of the epithelial cells and goblet cells. 41 A grading scheme was introduced in 1985 by Tseng, 42 but the more widely used grading scheme, and used in some CIC studies of contact lens wearers, was proposed by Nelson. 43,44 Other grading schemes are slightly different in that the number of steps or stages to the cellular changes is different.…”

Biomarkers of Ocular Surface Disease Using Impression Cytology

“…Introduced by Thatcher et al [1] and Egbert et al [2], IC is an easy-to-use technique of directly retrieving (mainly conjunctival) cells, using various filter types which are gently applied to exposed (interpalpebral) conjunctival surfaces. The filters used include cellulose acetate [3], polycarbonate (Biopore, Millipore UK Ltd, Watford, UK) [4], nitrocellulose [5] or polyethersulfone (PES) [6] which are gently applied to exposed (interpalpebral) conjunctival surfaces. Recently, a more standardized tool for IC sampling has been introduced (Figures 1 & 2).…”