Cameron Postnikoff is currently pursuing an MASc in Systems Design Engineering at the University of Waterloo, Canada.
Sodium fluorescein has been used as an ophthalmic dye and indicator of ocular surface health for over 100 years1. Fluorescein staining at the ocular surface has since been termed, “corneal staining,” but this staining usually presents itself as an appearance of fluorescent dots, known as punctate staining. Historically, sodium fluorescein staining has been thought to be a result of one of three mechanisms2: pooling in areas of shed cells, ingress around cells due to loss of tight junctions, or in dead or desquamating cells. The following is a review of a 2011 article by Mokhtarzedeh et al.3, which proved to contradict these historical theories.
Mokhtarzadeh M, Casey R, Glasgow BJ. Fluorescein punctate staining traced to superficial corneal epithelial cells by impression cytology and confocal microscopy. Invest Ophthalmol Vis Sci 2011;52: 2127-2135.
To evaluate fluorescein staining at a cellular level, the Glasgow group developed an impression cytology method whereby a membrane is trimmed to a very small size and is then pressed onto the cornea to remove epithelial cells, some of which were previously stained with fluorescein. The membrane is lifted off the cornea and processed to examine any cells that stuck to the membrane filter. Biomicroscopy is then performed to examine whether or not the punctate stains are still present on the ocular surface.
Optimization of methods
To optimize their impression cytology protocol, many different membranes were tested for their effectiveness in the removal of cells. In a small pilot study, Thinda et al. first compared mixed cellulose ester membranes to polycarbonate membranes and showed that while polycarbonate membranes may offer easier and cleaner post-processing, the polycarbonate membranes had a reduced cellular yield4. Mokhtarzedeh et al. then compared many different glass, polymeric, and cellulose membranes by first assessing their ability to remove cells from the buccal mucosa (inside of the cheek). Only membranes that showed high cellular yield were subsequently used on the cornea. For the cornea, polytetrafluorethylene (PTFE) membranes showed the greatest promise for the combination of cellular yield and ability to stain. The process of impression cytology using small PTFE membranes on the order of 2-3 mm has thus far proven to be safe and may be as exfoliative as a sampling using Schirmer strips (personal communication, Glasgow, June 2013).
Fluorescein was instilled onto the ocular surface and small areas of the corneal epithelium were removed by impression cytology, which allowed Mokhtarzedeh to make several observations related to staining:
- In most cases, punctate spots removed by impression cytology were traced from the cornea to a specific cell on the membrane. For a few spots, it was not possible to trace them to cells on the membrane, which may imply that there may be occasional spots of pooling. However, given that these non-traceable spots represented a minority, the absence of cells at the expected location on the membrane may also be due to the processing of the membrane.
- On the membrane, sodium fluorescein was localized to cells and not to the intercellular spaces; notably, there was negative staining in the ingress around cells on the cytology membrane. Confocal microscopy of fluorescein-stained cells on corneal transplants from patients with punctate keratopathy and dry eye also showed that the fluorescein was localized to the cytoplasm, and not to the nuclei of the cells.
- Sodium fluorescein staining was observed primarily in cells from the superficial layer, but fluorescein-stained cells could be found in up to the first three layers of corneal epithelium. Mokhtarzedeh et al. observed that if the membrane was applied for three to five seconds, a monolayer of cells would be removed. Prolonged membrane application of 20 to 30 seconds would subsequently remove multiple layers of cells. Fluorescein staining did not completely disappear from the cornea after a monolayer of cells was detached, but lifting multiple epithelial cell layers was able to remove all punctate staining on the cornea. This implies that superficial cells are not solely responsible for the corneal staining response.
The mechanism of staining
While Mokhtarzedeh’s results were obtained on patients with dry eye, the results provide new evidence on which cells are actually staining with fluorescein and how they correspond to the punctate dots that are observed at the slit lamp. This study provides evidence that the classical mechanisms proposed to describe corneal staining (pooling, ingress, and dead cells) do not accurately represent sodium fluorescein staining. Dr. Maud Gorbet’s editorial outlines some of the in vitro and ex vivo investigations that have been undertaken to better understand the mechanisms of solution-induced corneal staining (SICS). These cell studies corroborate Mokhtarzedeh’s observations and imply that the mechanism of fluorescein staining may be similar between dry eye and SICS. However, it has yet to be determined what causes corneal epithelial cells to stain with sodium fluorescein. In any case, corneal staining is not limited to a superficial epithelial layer response, as a contribution of fluorescein-stained cells can be found deeper into the corneal layers in dry eye patients.
With continued improvement of the understanding of the biological and cellular processes of sodium fluorescein transport, fluorescein interaction with the cornea can be much better understood. Ultimately, this will enable us to better contextualize corneal staining and its link to biocompatibility.
I would like to sincerely thank Dr. Ben Glasgow from UCLA for the insightful discussion on his group’s research.
1. De Schweinitz GE. Diseases of the Eye: A Handbook of Ophthalmic Practices. 1st ed. Philidelphia, PA: W.B. Saunders; 1893.
2. Morgan PB, Maldonado-Codina C. Corneal staining: Do we really understand what we are seeing? Cont Lens Anterior Eye 2009;32: 48-54.
3. Mokhtarzadeh M, Casey R, Glasgow BJ. Fluorescein punctate staining traced to superficial corneal epithelial cells by impression cytology and confocal microscopy. Invest Ophthalmol Vis Sci 2011;52: 2127-2135.
4. Thinda S, Sikh PK, Hopp LM, Glasgow BJ. Polycarbonate membrane impression cytology: evidence for fluorescein staining in normal and dry eye corneas. Br J Ophthalmol 2010;94: 406-409.