BritishJournal ofOphthalmology, 1990,74,519-522 519 Staining of the conjunctiva and conjunctival tear Br J Ophthalmol: first published as 10.1136/bjo.74.9.519 on 1 September 1990. Downloaded from film Joseph A Eliason, David M Maurice Abstract reduces the contrast between the surface and the Both the distribution of tear fluid over the background. conjunctiva and any injury to the conjunctival It was thought possible that problem (3) could epithelium can be made visible by instilling a be overcome by the use ofa more hydrophilic dye fluorescent solution into the eye and observing such as carboxyfluorescein, but it was decided to with an appropriate combination of excitation evade problem (2) as well by using the fluoro- and barrier filters. Sulphorhodamine B, which phore, sulphorhodamine B, which is not only has an orange fluorescence that can be sepa- very hydrophilic but can be excited by wave- rated from the green natural fluorescence of lengths too long to lead to visible fluorescence in the ocular tissues, gives a greater contrast than the tissues. When observed with an appropriate fluorescein. The tear film is seen to cover the pair ofcolour filters, it allowed both the tear film surface of the conjunctiva and to be concen- and devitalised confunctival cells to be readily trated in its folds. Small circular areas of thin observed with high contrast against the tissue tear film appear transiently in the neighbour- background. hood of the limbus after a blink. Occasional The purpose of this report is to emphasise the cells stain on the normal conjunctiva, partic- advantages ofan appropriate pair ofcolour filters ularly in the interpalpebral area. The density of when fluorescent dyes are used to examine the the staining increases in dry eye conditions. conjunctival surface, and to illustrate the Conjunctival trauma is sensitively revealed by additional benefit of using a dye with larger the method, and its healing can be followed. wavelength fluorescent characteristics than Hard contact lenses are seen to traumatise fluorescein. In addition, we note some hitherto continually the inferior limbal conjunctiva in undescribed physiological and anatomical prop- symptomless wearers. erties of the conjunctival tear film and survey some clinical conditions in which the technique might prove to have value. (These observations The instillation of a drop of fluorescein is a have already been briefly noted in the abstracts of common method of detecting injury to the the First Meeting of the International Society of corneal epithelium. Before damaged cells are Dacriology, Budapest 1987, and ARVO 1988.) revealed by the stain, the precorneal tear film is http://bjo.bmj.com/ coloured by the dye, and it is possible to examine it for normal variations in uniformity or for the Materials and methods appearance ofdark patches when the layer breaks Sulphorhodamine B (molecular weight 559) is similar in up. The conjunctival cells and the precon- chemical structure and properties to fluorescein but is less junctival tear film may be of equal physiological lipid soluble, with an octanol/water partition coefficient of interest to that of the corneal 0-0033 compared with 0-61 for fluorescein.' Its fluores- and pathological cence is orange and is most readily excited by green light, epithelium, but no equivalent method ofstaining the peak absorption and emission wavelengths being 556 on September 28, 2021 by guest. Protected copyright. them has been available. Rose Bengal is and 572 nm. It is usually supplied as an acid and must be employed as a stain for conjunctival damage, but dissolved in alkali, being soluble to the extent of about it provides poor contrast as usually applied, and 20 g/l at pH 7. The toxicity ofthe compound appears to be is uncomfortable when instilled in the eye. very low. Doses of up to 1 g/kg could be injected intraperitoneally in the mouse without causing death, and There are three reasons why staining with 50 [d ofthe saturated solution injected into the stroma ofa fluorescein has not been found valuable over the rabbit cornea had as little effect as an equal volume of conjunctival surface: saline. (1) The use of an inefficient optical filter system. The subjects were patients, residents, and staff of the Generally, a cobalt filter has been placed in front ophthalmology clinic. Informed consent was obtained of the slit-lamp light and the eye has been after the nature ofthe procedure had been fully explained. A 0-5% solution of the dye at pH 7, made 300 mM with observed under the microscope without the sucrose, was used to stain the tear film and a small drop intervention of a barrier filter. This is adequate was applied to the tarsal conjunctiva with a wooden for the observation over the dark background of applicator. The solution did not cause discomfort in the the cornea, but the scattering ofthe incident blue eye of any volunteer or patient. It is not important, Department of light by the sclera hides the weaker fluorescent however, to adhere exactly to this dye formulation. Ophthalmology, Stanford light emitted by the dye. The eye was observed with a Haag-Streit 900 slit-lamp University Medical using a broad patch of illumination. A band-pass inter- Center, Stanford, USA (2) The background fluorescence of the tissue. ference filter of 520-550 nm, such as is used as a barrier J A Eliason Even with an efficient pair of excitation and filter for fluorescein, was inserted in the illuminating light D M Maurice barrier filters the blue incident light will excite a path, and gelatin absorption filters (Kodak No 22) Correspondence to: greenish fluorescence in the conjunctiva and transmitting 100% of the light at 600 nm were placed in D Maurice, Department of Ophthalmology A157, sclera, which is seen as a general glow that the eyepieces. This combination offilters passes less than Stanford University Medical reduces the contrast of the fluorescein. 0-01% of light at the cross-over point of their absorption Center, Stanford, CA 94305, curves, around 570 nm. Photographs were taken with a USA. (3) Penetration of fluorescein. Because of the Zeiss photoflash slit-lamp on 1600 ASA colour film with Accepted for publication slight lipid solubility of the dye, it can stain the the same combination of filters. Black-and-white film of 15 March 1990 conjunctiva tissue appreciably, and this further similar speed can also be used. 520 Eliason, Maurice Br J Ophthalmol: first published as 10.1136/bjo.74.9.519 on 1 September 1990. Downloaded from Figure I Staining oftear film with sulphorhodamine B in a normal subject. ((A) Pattern over conjunctiva immediately after a blink. (B) Same area of conjunctiva a few seconds later. Note the enhancement oflines and patches with time. Figure IA Figure IB fluid rather than tissue staining, since they disappeared after thorough flushing with saline or on pressing the conjunctiva under a clear plastic plate (Fig 2). Many features of the patterns of the lines remained constant over a long period, and they almost certainly corres- pond to folds in the conjunctiva (Figs 3A, B). The wider patches may represent layers ofmucus or broad depressions in the conjunctival surface. The thickness of the mucus layer on the con- Figure 2 Disappearance junctiva has been reported to vary over the ofconjunctival pattern with surface.2 gentle pressure from a transparent plastic plate Some transient phenomena occur after a blink. (arrow). The tear film over the entire cornea and con- junctiva appeared to flow upwards immediately Results after the opening of the eye. This movement lasted for about 3 seconds. Often circular dark NORMAL TEAR FILM patches about 1 mm in diameter were observed in In the absence of the dye the tissues of the eye the tear film over the conjunctiva and cornea, appeared black through the microscope, making most frequently near the limbus (Fig 4). They focusing on the ocular surface difficult. Pin- appeared immediately after a blink, flowed gueculae gave rise to a natural fluorescence which upward with the tear film, and vanished in a http://bjo.bmj.com/ was absent in normal sclera or cornea. second or two by constricting to smaller dia- After the dye was instilled the precorneal tear meters. The origin of these spots has not been film and the lacrimal strips shone as brilliantly as examined, but it seems most likely that they they do with fluorescein under blue illumination. would correspond to an area where the aqueous Furthermore, the conjunctiva was seen, in the layer is thinner rather than where it is diluted. first moments after instillation, to be covered with a more or less uniform tear film. Shortly on September 28, 2021 by guest. Protected copyright. thereafter a system oflines and patches appeared CONJUNCTIVAL STAINING which became more prominent during the first If the dye was removed from the tear film by seconds immediately after opening the eye, while thorough flushing with saline, or ifit was allowed the fluorescence ofthe tear film covering the area time to be washed out in the normal tear secre- between them became weaker (Figs 1A, B). tions, the background that remained was either These seem to be a result of accumulations of dark or showed a faint orange glow from dye that Figure 3 Staining with sulphorhodamine B in a normal subject. (A) Pattern ofconjunctival lines. (B) Same area ofconjunctiva after an interval ofa year. Arrows indicate some ofthe similarities. Figure 3A Figure 3B Staining ofthe conjunctiva and conjunctival tearfilm 521 constant on retaining the tissue. They probably Br J Ophthalmol: first published as 10.1136/bjo.74.9.519 on 1 September 1990. Downloaded from correspond to individual epithelial cells that are either damaged or in a permeable condition before desquamation.