Name: impression cytology of the lid wiper area
Uploaded: 2016-08-09T15:00:00
Description: Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

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.

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.
<ol>
	<li>Add 5 &#181;l Ethidium (or 4 &#181;M) and 5 &#181;l Calcein AM (or 4 &#181;M) 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.</li>
</ol>
2. Collect Cells
<ol>...

<ol>
	<li>Barbato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+variation+in+spontaneous+eye-blink+rate.">Diurnal variation in spontaneous eye-blink rate.</a> Psychiatry Res. 93, 145-151 (2000).</li><li>Korb, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lid-Wiper+Epitheliopathy+and+Dry-Eye+Symptoms+in+Contact+Lens+Wearers.">Lid-Wiper Epitheliopathy and Dry-Eye Symptoms in Contact Lens Wearers.</a> Eye &amp; Contact Lens. 28, 211-216 (2002).</li><li>Dumbleton, K., Woods, C. A., Jones, L. W., Fonn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+contemporary+contact+lenses+on+contact+lens+discontinuation.">The impact of contemporary contact lenses on contact lens discontinuation.</a> Eye &amp; Contact Lens. 39, 93-99 (2013).</li><li>Roba, M., Duncan, E., Hill, G., Spencer, N., Tosatti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+measurements+on+contact+lenses+in+their+operating+environment.">Friction measurements on contact lenses in their operating environment.</a> Tribol Lett. 44, 387-397 (2011).</li><li>Sickenberger, W., Pult, H., Sickenberger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIPCOF+and+contact+lens+wearers:+a+new+tool+to+forecast+subjective+dryness+and+degree+of+comfort+of+contact+lens+wearers.">LIPCOF and contact lens wearers: a new tool to forecast subjective dryness and degree of comfort of contact lens wearers.</a> Contactologia. 22, 74-79 (2000).</li><li>Pult, H., Purslow, C., Berry, M., Murphy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+tests+for+successful+contact+lens+wear:+relationship+and+predictive+potential.">Clinical tests for successful contact lens wear: relationship and predictive potential.</a> Optom Vis Sci. 85, E924-E929 (2008).</li><li>Doughty, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+features+of+cells+along+Marx's+line+of+the+marginal+conjunctiva+of+the+human+eyelid.">Morphological features of cells along Marx's line of the marginal conjunctiva of the human eyelid.</a> Clin Exp Optom. 96, 76-84 (2013).</li><li>Jalbert, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+human+lid+margin+epithelium+using+impression+cytology.">Assessing the human lid margin epithelium using impression cytology.</a> Acta Ophthalm. 90, e547-e552 (2012).</li><li>Nelson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impression+cytology.">Impression cytology.</a> Cornea. 7, 71-81 (1988).</li><li>Egbert, P. R., Lauber, S., Maurice, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+conjunctival+biopsy.">A simple conjunctival biopsy.</a> Am J Ophthalmol. 84, 798-801 (1977).</li><li>Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Confocal microscopy. , 1-64 (1990).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Tomlins, P., Roy, P., Curnow, J., Rauz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+EyePRIM+Device+for+Conjunctival+Impression+for+Flow+Cytometry.">Assessment of the EyePRIM Device for Conjunctival Impression for Flow Cytometry.</a> Invest Ophthalm Vis Sci. 54, 5430-5430 (2013).</li><li>Knop, E., Korb, D. R., Blackie, C. A., Knop, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lid+margin+is+an+underestimated+structure+for+preservation+of+ocular+surface+health+and+development+of+dry+eye+disease.">The lid margin is an underestimated structure for preservation of ocular surface health and development of dry eye disease.</a> Res Proj Dry Eye Syn. 45, 108-122 (2010).</li><li>Knop, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lid+wiper+and+muco-cutaneous+junction+anatomy+of+the+human+eyelid+margins:+an+in+vivo+confocal+and+histological+study.">The lid wiper and muco-cutaneous junction anatomy of the human eyelid margins: an in vivo confocal and histological study.</a> J Anat. 218, 449-461 (2011).</li><li>Christensen, J. A., Skaarland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+cell+area+measurements+in+the+cytological+evaluation+of+pleural+effusions:+a+study+of+subjective+assessments+and+morphometric+measurements.">Nuclear and cell area measurements in the cytological evaluation of pleural effusions: a study of subjective assessments and morphometric measurements.</a> Diag Cytopathol. 3, 50-54 (1987).</li></ol>

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 ...

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 &#34;lid wiper&#34; 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 lens material and the ocular surface has been shown to change4. There is evidence to suggest that dryness symptoms could be related to this altered friction2,5,6.
This association may also be reflected in clinically observable phenomena, notably lid wiper epitheliopathy (LWE), also called upper lid margin staining (ULMS)6. LWE is an early sign of ocular irritation and a potential predictor for dry eye disease. It is observed and measured by the vital staining of the upper and lower lid margin regions. While this grading system2 and its clinical validity (i.e., correlation with subjective symptoms) are still under debate, ocular discomfort remains a conundrum for clinicians and scientists alike and, most importantly, an inconvenience for patients.
To-date, little is known about clinically-relevant variations in the (sub-)cellular anatomy and physiology of the lid wiper area7 and only one report makes use of cytological methods8. Impression cytology (IC) has been employed for over 40 years to collect cells from the epithelial surface of the bulbar or tarsal conjunctiva by application of a membrane9,10. Upon removal, the adherent cells undergo cytological staining and microscopic imaging. Due to the anatomic and cellular differences in the lid wiper region, this technique requires optimization.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

impression cytology of the lid wiper area

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 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 ...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

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 ...

The overall goal of this procedure is to collect, process and visualize epithelial cells from the eyelid margins to assess the effects of friction at the ocular surface. This method may help answer key questions about ocular discomfort, particularly in patients with dry eye and contact lens related discomfort. The main advantages of this technique are that it is relatively simple, quick, minimally invasive and may be used to assess the effects of friction on ocular epithelial cells.Begin by removing the membrane inserts from their package. Using a permanent marker, label the inserts for each eyelid to be sampled, marking the orientation of the cell collection on the side of the plastic holder of the membrane. Next, dispense one drop of topical anesthetic into the lower conjunctival sac of each eye and instruct the participant to keep both eyes closed for one minute.Then, evert one upper eyelid by the lashes, avoiding contacting with the lid margin, and lightly apply the insert membrane perpendicularly onto the central part of the lid wiper region. After three to five seconds, carefully remove the membrane and ask the participant to look up to allow the lid to flip back into place. Immediately apply one drop of PBS to hydrate the sample.Then, apply ocular lubricants and instruct the participant to avoid eye rubbing for the rest of the day. Immediately after cell collection, use micro-scissors to cut the membrane in half perpendicularly through the middle of the cell collection line. Then, cut along the outer margin of one membrane half to separate it from the plastic holder.Before completely detaching the membrane from the holder, secure the sample with tweezers and transfer the liberated membrane into a labeled two milliliter centrifuge tube containing 500 microliters of 95%ethanol for 20 to 120 minutes. For immunocytochemical staining of the sample, separate the second half of the membrane from the holder and carefully place it in a 35 millimeter glass bottom culture dish with the collection side facing down. Next, add 20 microliters of freshly prepared immunocytochemical stain to the membrane.Use disposable pipette tips to carefully push the membrane edges down as necessary, and gently cover the sample with a glass cover slip. Cover the dish and seal the edges of the dish with lab film. Then, label the dish with a lab marker and promptly proceed with imaging of the sample.For cytological staining, begin by hydrating the membrane. Then, stain the sample according to standard staining protocols. After completing the staining procedure, transfer the sample onto a glass slide, aligning the cell collection line with a slide margin for easier imaging.Then apply a drop of 100%ethanol to the membrane and cover the sample with a glass slip. To image the immunocytochemically stained sample, select the lowest magnification on the appropriate microscope for visualizing the stain and place the sample on the microscope stage. Activate the digital imaging system and inspect the cells on the computer monitor for the cellular features of interest.Then, select the appropriate microscope magnification and acquire images with the microscope software. To image the cytologically stained sample, mount the slide onto a bright field microscope stage. At the microscope's lowest magnification, ensure that the cell collection is aligned with the appropriate axis of the microscope stage control.To adjust the sample, remove the cover slip and use tweezers to rotate the membrane. Rehydrate the sample with 100%ethanol, then capture images of the entire sample at the lowest magnification. At the 10 to 20X magnification, adjust the microscope light source and software imaging parameters, and disable the automated software metering.Then, determine the beginning and endpoints of the scan and capture images of the entire cell collection within these points. Fluorescent immunocytochemical dyes reveal the esterase activity of the lid wiper collected cells, indicative of the cell viability as well as the presence of nucleic acids in the sample, reflecting a compromise of the conjunctival cell membranes. Cytological staining facilitates the fine distinction between the degrees of keratinization in the epithelial cells.For example, here the transition between a low and advanced keratinization in the tarsal conjunctiva through the lid wiper conjunctiva and the mucocutaneous junction respectively can be observed. Panoramic imaging of the cytologically stained cell collections permits an analysis of the entire collection area, with an image resolution adequate for zooming to the nuclear level. Once mastered, this technique can be completed in about two hours if it is performed appropriately.While attempting this procedure, it is important to remember that a correct angle and pressure of the membrane application are essential. Some experience is required to obtain consistent, repeatable results. Following this procedure, future clinical trials should explore the cellular particularities of contact lens wearers and subjects with lid wiper epitheliopathy, dry eye or dryness symptoms, to shed further light on the topic of ocular discomfort.

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