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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Impression Cytology of the Lid Wiper Area

Published: August 9th, 2016

DOI:

10.3791/54261

1Centre for Contact Lens Research, University of Waterloo

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. 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 LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

The human upper eyelid executes around 10,000 blinks every day1, with just a 1 - 2 mm narrow conjunctival region opposing the ocular globe. Due to its wiping motion during the blink, this portion of the lid margin has been termed the "lid wiper" region2. It is assumed that friction is increased in this region during habitual blinking, due to the lid wiping over the globe. This may play a significant role in ocular comfort. Contact lens wearers in particular experience ocular discomfort and this is one of the primary reasons for ceasing lens wear3. When a contact lens is placed on the eye, the friction coefficient between the le....

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Ethics statement: Prior to collecting cells from human subjects, informed consent and ethics approval must be obtained.

1. Prepare Stain

NOTE: Prepare stain on day of experiment.

  1. Add 5 µl Ethidium (or 4 µM) and 5 µl Calcein AM (or 4 µM) to 2.5 ml phosphate buffered saline (PBS) in 15 ml centrifuge tube; mix. Wrap in aluminum foil to shield from light and store at RT until use.

2. Collect Cells

    .......

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The cell culture membrane spanned over the plastic holder is a convenient tool for handheld collection of epithelial cells from the lid wiper conjunctiva. This method eliminates the need for additional sterilized instruments typically used to prepare and handle IC membranes. Figure 1 depicts the IC of the lid wiper area using a cell culture insert. We optimized two different staining protocols that complement each other to characterize epithelial cells from the lid wiper .......

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Impression cytology is typically performed on the bulbar conjunctiva, using mixed cellulose ester membranes. Samples are cut to size from bulk material sheets, sterilized, applied and removed from the conjunctival surface using forceps. Using the same membrane material, a piston-controlled IC device was recently designed to match the surface geometry of the bulbar conjunctiva, and maintain consistent pressure between applications13. These approaches are inefficient for the narrow, prominently curved lid wiper .......

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We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

....

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Name Company Catalog Number Comments
Alcian Blue, 1% in 3% Acetic Acid Sigma B8438-250ML
Hematoxylin, Gill 1 Sigma GHS116-500ML
Papanicolaou stain, OG-6 Sigma HT40116-500 ml
Papanicolaou stain, Modified EA Sigma HT40232-1L
Live/Dead Viability/Cytotoxicity Kit Life Technologies L-3224 contains ethidium homodimer-1 and Calcein AM dyes
Alcaine (0.5% proparacaine hydrochloride) Alcon
Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm EMD Millipore PICM01250 
35 mm Glass Bottom Dishes MatTek Corporation P35G-0-20-C

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  13. Tomlins, P., Roy, P., Curnow, J., Rauz, S. Assessment of the EyePRIM Device for Conjunctival Impression for Flow Cytometry. Invest Ophthalm Vis Sci. 54, 5430-5430 (2013).
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  15. Knop, E., et al. The lid wiper and muco-cutaneous junction anatomy of the human eyelid margins: an in vivo confocal and histological study. J Anat. 218, 449-461 (2011).
  16. Christensen, J. A., Skaarland, E. Nuclear and cell area measurements in the cytological evaluation of pleural effusions: a study of subjective assessments and morphometric measurements. Diag Cytopathol. 3, 50-54 (1987).

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