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.
