Impression Cytology of the Lid Wiper Area

Summary

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.

Abstract

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.

Introduction

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 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 friction^2,5,6.

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 membrane^9,10. 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.

Protocol

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

1. Remove cell culture inserts from their package and label for each eyelid to be sampled. Mark orientation of cell collection on the side of the membrane plastic holder using permanent marker. Conduct slit lamp inspection at moderate magnification (approximately 20X) to confirm appropriate health of the region to undergo IC.
2. Dispense one drop of topical anesthetic (0.5% proparacaine hydrochloride) in the lower conjunctival sac of each eye and instruct participant to keep eyes closed for one minute.
3. Evert upper eye lid and hold in place by the lashes; avoid any contact with the lid wiper area.
   Optional: the aid of a secondary investigator may be required to perform this step.
4. Apply membrane perpendicularly onto the central part of the lid wiper region with minimal pressure.
5. Hold membrane in place for 3 - 5 sec. Observe the membrane become translucent in the contact area. At this point, gently remove the membrane and permit the participant's lid to "flip" back into place.
6. Promptly apply one drop of PBS to the sample, to prevent it from drying out. Membrane will turn translucent and should not turn opaque at any time, as this indicates drying. Have the secondary investigator proceed immediately with processing of samples (section 3).
7. Conduct final eye check to ensure integrity of the conjunctiva. Dispense ocular lubricants and instruct participant to avoid rubbing their eyes until anesthetic effect wears off (approx. 15 min).

3. Sample Processing

1. Using micro-scissors, cut the membrane in half (one half for each of the downstream analyses), perpendicular through the middle of the cell collection. Cut along the outer margin of one membrane half to separate it from the plastic holder; avoid interaction with the collected cells on the membrane, including ensuring that the membrane does not fold onto itself.
2. Secure the membrane using tweezers before completely detaching from holder. Place membrane into labelled 2 ml centrifuge tube containing 500 µl of 95% (v/v) ethanol and leave to fix for a minimum of 20 min to a maximum of 2 hr before processing (section 4.2).
3. Immediately separate second half of membrane (as described in section 3.2) and promptly proceed with section 4.1.

4. Sample Staining

1. Immunocytochemical Staining
   1. Carefully place membrane in 35 mm glass bottom culture dish with collection side facing down; ensure membrane is flat.
   2. Pipette 20 µl of immunocytochemical stain composition assembled in section 1 on to the membrane. If sample curls up, use disposable pipette tips to carefully flatten the membrane by pushing its edges down. Avoid contact with cell collection areas.
   3. Gently cover the membrane with glass cover slip. Avoid unnecessary movement of sample. Cover dish with lid and seal with lab-film around the edges. Do not obstruct transparency of dish (top and bottom) and visibility of sample. Label with lab marker.
   4. Immediately proceed with imaging (section 5.1), then return to section 4.2 to continue with cytological staining of the other half of the membrane.
2. Cytological Staining
   1. Gradually hydrate membrane by slowly adding 500 µl of distilled water to the tube used for fixation in section 3.4. Aspirate contents several times into pipette tip to mix, then remove contents.
   2. Add 500 µl of distilled water. Let stand for a few seconds, then remove. Add 500 µl of Alcian Blue stain and leave for 3 min. Remove stain and perform 3 consecutive 500 µl distilled water rinses or until liquid rinses clear.
   3. Add 500 µl of Hematoxylin # 1 stain and leave for 3 min. Remove stain and perform three consecutive 500 µl distilled water rinses or until liquid rinses clear. Dehydrate (reverse of section 4.2.1) by aspirating the clear liquid.
   4. Add 500 µl of Papanicolaou OG-6 stain and leave for 3 min. Remove stain and perform one 500 µl 95% (v/v) ethanol rinse.
   5. Add 500 µl of Papanicolaou EA-65 stain and leave for 3 min. Remove stain and perform 3 consecutive 500 µl 95% (v/v) ethanol rinses.
   6. Fully dehydrate using three consecutive 500 µl 100% ethanol rinses. Using tweezers, remove sample from solution; avoid touching cell collection areas.
   7. Place membrane on glass slide; ensure that cell collection line is either parallel or perpendicular to slide margin for easier alignment during imaging.
   8. Apply a drop of 100% ethanol and cover with glass slip. Immediately proceed with imaging (section 5.2).

5. Sample Imaging

1. Imaging Fluorescent Immunocytochemical Stains
   1. Use a confocal laser scanning microscope (CLSM)¹¹ or fluorescence microscope¹², equipped with a digital color camera, connected to a computer running an image acquisition software. Due to the samples being contained within culture dishes, an inverted microscope may be necessary.
   2. Set up microscope^11,12 according to absorption and emission spectra of ethidium homodimer-1 (528/617 nm Ex/Em maxima in the presence of DNA) and calcein AM (494/517 nm Ex/Em maxima).
   3. Select the microscope's lowest magnification (e.g., 2.5X objective) and activate the digital imaging system; observe the sample displayed on the computer monitor.
   4. Inspect sample for cellular features of interest.
   5. Select desired microscope magnification and acquire images using imaging software.
   6. Save files in either native microscope file format or an uncompressed file format (e.g., *.raw, *.tiff).
2. Imaging Cytological Stains
   1. Use a standard laboratory bright-field microscope with no filters, equipped with a digital color camera, connected to a computer running an image acquisition software.
   2. Place and lock glass slide (from step 4.2.8) on microscope stage. Rehydrate sample with 100% ethanol as needed, throughout the entire imaging procedure.
   3. Select the microscope's lowest magnification (e.g., 2.5X objective) and activate the digital imaging system; observe the sample displayed on the computer monitor.
   4. Ensure cell collection is aligned with either X or Y axis of microscope stage control. Adjust if needed, by removing the cover slip and rotating the sample using a pipette tip or tweezers.
   5. Using the imaging software, capture image(s) of the entire sample with the lowest microscope magnification.
   6. Switch to moderate microscope magnification (e.g., 10 - 20X objective) and adjust microscope light source and software imaging parameters (exposure, contrast, white balance, etc.), and disable their automated software metering. Save these settings for future use.
   7. Determine beginning and end points of scan (e.g., top left and bottom right corners of cell collection).
   8. Using the imaging software, capture image(s) of the entire cell collection area within the starting and end points. Ensure a 20% side-to-side and top-to-bottom overlap between all adjacent images.
   9. Save files using the native microscope file format or an uncompressed file format (e.g., *.raw, *.tiff).
   10. Generate final panoramic image using automated stitching software of choice (e.g., Adobe Photoshop Elements). Use reference images taken at step 5.2.5) to confirm the accuracy of the automated stitching in the final image.

Results

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 ...

Discussion

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 ...

Materials

List of materials used in this article

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