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Alex Muntz, Kevin van Doorn, Lakshman N Subbaraman, Lyndon William Jones, ; Impression cytology of the lid wiper area. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4432.
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© ARVO (1962-2015); The Authors (2016-present)
Few reports on the cellular anatomy of the lid wiper (LW) area exist and only one makes use of cytological methods. Impression cytology (IC) is typically performed on bulbar or tarsal conjunctiva and thus requires optimization for use on the LW. The purpose of this study was to optimize a method of collecting, staining and imaging cells from the LW region using IC.
Upon anesthesia (Alcaine, 0.5%), the upper lids of 5 subjects (n=10) were everted and IC was conducted using various membranes (MF-Millipore, PTFE Biopore Millipore, Opia EYEPRIM). Several fixatives (100% Methanol, 95% Ethanol), histological stains (Papanicolaou (hematoxylin GILL-1, OG-6, EA-65), Periodic Acid-Schiff (PAS) and Alcian Blue (AB)) and soak times (1,3,5 minutes) were tested. Varying concentrations of fluorescent dyes (Annexin V, Calcein AM, Ethidium homodimer-1) were tested and imaged using confocal laser scanning microscopy (CLSM).
IC delivered optimal results when using the PTFE Biopore membrane. Fixing in 95% Ethanol for >20 minutes then staining in 500µl each of AB, GILL-1, OG-6 and EA-65 for 3 minutes revealed the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Calcein AM (4µM), Ethidium (4µM), Annexin V (5µl in 250µl buffer) were combined to successfully show metabolic activity, compromised cell membranes, nucleic acids and apoptosis in cells. Up to 200 microscopy digital images were captured for each sample and stitched into a high-resolution, large scale image of the entire IC span.
We have developed a protocol consisting of an optimal selection of membrane, stains and imaging procedures and successfully showed that the sensitivity of IC is appropriate for identifying distinct cellular morphologies surrounding the LW area, as well as showing varying degrees of metabolic activity. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the lid wiper area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.
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