NR 1/2015 ART.112

Lek. wet. Aleksandra Tomkowicz

Division of Small Animal Surgery and Anaesthesiology,
Department of Small Animal Diseases with Clinic, Warsaw University of Life Sciences.

lek. wet. Jan Frymus

Division of Small Animal Surgery and Anaesthesiology,
Department of Small Animal Diseases with Clinic, Warsaw University of Life Sciences.

lek. wet. Piotr Kowalczyk

Division of Small Animal Surgery and Anaesthesiology,
Department of Small Animal Diseases with Clinic, Warsaw University of Life Sciences.

prof. dr hab. Marek Galanty

Division of Small Animal Surgery and Anaesthesiology,
Department of Small Animal Diseases with Clinic, Warsaw University of Life Sciences.

Fluorescein is an orange dye widely used in both human ophthalmology and veterinary, it is available as a 2% eye drops or disposable strips. Fluorescein has been used in the diagnosis of corneal ulcers, obstruction of the nasolacrimal duct (Jones test), disorders of the tear film quality (TBUT- tear breakup time) or perforation of the eyeball (Seidel test). It is easy to use, low cost, and allows for quick diagnosis. Since 1961 is also used in fluorescein angiography. In veterinary medicine, posterior segment angiography is usually performed, it can shows tumors, inflammation or degenerative processes. Without a doubt, disadvantage of posterior segment angiography is expensive equipment that is required.

Key words: fluorescein, ocular examination, retinal angiography

Fluorescein is an orange-colored dye, widely used in ophthalmology. It was first synthesized by Baeyer in 1879, three years later, in 1882, when researchers discovered that fluorescein has the ability to reveal corneal defects (20), it was used on the eye for the first time.

From the chemical point of view, fluorescein is a water-soluble weak dibasic acid of the xanthene group. Sodium fluorescein is the most commonly used form of fluorescein, it converts almost 100% of absorbed light with a peak wavelength of 520 nm, at acid pH appears orange, after contact with tears, green.

Fluorescein is now used in various areas of veterinary ophthalmology to detect corneal ulcers, corneal perforations, qualitative dry eye syndrome and nasolacrimal system occlusions (4, 19, 23, 28). It is also used in retinal vasculature imaging and in research as a systemic marker in pharmacokinetic and pharmacology studies.

Fluorescein is available as multi-dose 2% ophthalmic drops or as a single-dose paper strip. Use of a sterile single dose paper strip is recommended in order to prevent transmission of bacterial and viral pathogens via contaminated ophthalmic solutions. Storey has reported that feline coronavirus (FCV) remained infectious in sodium fluorescein solution for up to 7 days (29). FCV may be recovered from eyes of cats with clinical signs such as fever, rhinitis, glossal ulceration and conjunctivitis. Also ocular bacterial infection caused by Pseudomonas aeruginosa after use of multidose solution has been reported in the dog. In case of use of drops a new bottle should be opened every month. Because dye may cause discomfort and irritation, it may be necessary to apply one drop of local anesthetic (Proxymetacaini hydrochloridum) and wash the residue from conjunctival sac after ocular examination.

Ulcerative keratitis

Cornea is a transparent front part of the eye. It is a fibrous tunic consisting of corneal epithelium, corneal stroma, Descement’s membrane and corneal endothelial cells. During ophthalmic examination one drop of fluorescein solution is applied into the conjunctival sac or a fluorescein impregnated strip is moistened with eyewash or sterile saline and then touched to dorsal bulbar conjunctiva (4) (fot. 1).

Fot. 1 Fluorescein application; strip is moistened with sterile saline and then touched to dorsal bulbar conjunctiva.

After application, the eyelids should be closed or the animal should be allowed to blink to distribute dye on the ocular surface. Fluorescein, which is lyophobic and hydrophilic in healthy eye, does not stain corneal surface. In case of wounds that are deeper than basement membrane of the epithelium fluorescein stains the stroma green. Wounds not penetrating the epithelium basal membrane can be dyed with rose Bengal (8). Any exposed stroma, which is hydrophilic, absorbs dye, it is well visible in blue cobalt light although usually it is not necessary to diagnose the ulceration. Fluorescein does not stain a Descement’s membrane so ulcers reaching this layer have a characteristic appearance; the dye uptake occurs only on the walls of the wound (fot.2).

Fot. 2 Descementocele, dye uptake occurs only on the walls of the wound.

Ulcerative keratitis is one of the most common ocular diseases in small animals, it is suspected when lacrimation, blepharospasm, photophobia, corneal edema and conjunctival hyperemia are present.

The Jones test

Fluorescein dye passage is the primary test of patency. Excessive tearing and ocular discharge may be results of epiphora, nasolacrimal pump failure or reflex hypersecretion due to nasolacrimal duct constriction or obstruction (3). Other clinical signs are conjunctivitis, medial canthal swelling, draining fistula, alopecia and dermatitis. The procedure involves placing fluorescein on the conjunctiva and, after several minutes have elapsed, on the nasal area on the ipsilateral side (fot3)

Fot. 3 Positive Jones test, fluorescein visible on the nasal area after administration to the eye.

In brachycephalic animals, because of V shape nasolacrimal duct, dye can appears in pharynx. This occurs also in nonbrachycephalic breeds, approximately 40% of dogs have an additional communication between the duct and the oral cavity. The transit time is very variable and is significantly higher in brachycephalic and large animals (up to 20 minutes in horses) (10). Negative Jones test in brachycephalic breeds is not synonymous with duct obstruction; in these animals it has no diagnostic value. To properly perform Jones test in large animals (horses, cria, cattle), the required volume of fluorescein is a minimum of 3 milliliters (19). To obtain the required volume of solution, an impregnated single use paper strip is loaded into the syringe and mixed with sterile saline or eyewash. A positive result of Jones test (dye on the nasal area) is definitive for a patent duct but it does not mean that there is no anatomical abnormalities. A negative Jones test is only suggestive of a problem, Because of the possibility of false negative results further evaluation is required. Usually nasolacrimal flushing, contrast radiographs or computer tomography is indicated.

BUT- breakup time

The BUT break up time (or TBUT- tear break up time) is a noninvasive ocular diagnostic test that allows for an assessment of quality of precorneal tear film (PCF) by observing its stability on corneal surface (7, 9, 18). Tear film instability results from qualitative or quantitative tear film abnormalities and can be associated with ocular surface disease. Any tear film abnormalities can cause a dry eye syndrome, which is defined as a tear film disorder caused by tear deficiency, extreme tear evaporation or qualitative impairment, which leads to injury of the ocular surface. To diagnose tear deficiency, Schirmer test should be performed; in order to diagnose quantitative dry eye syndrome (5), the break up time is the preferred option. The BUT is a measure of stability of the precorneal film that involves recording the time it takes for fluorescein dye, and hence the tear film, to evaporate from the corneal surface. Tear BUT is thus an indirect measure of the mucin and/or lipid components of PCF. To perform TBUT, one drop of fluorescein dye should be applied into the eye, then the eyelid should remain closed to disperse dye on the surface of the eye. Timing begins when the eyelids are open again. The dorsolateral cornea is observed with the use of an ophthalmoscope or a slit lamp (cobalt blue light). Timing stops when the first sign of tear breakup – a dark area in the green fluorescent tear film – is noted. In the dog the mean TBUT is 19,7±5 seconds and in the cat 16,7±4,5 seconds (10). It is important to keep in mind that the ranges are broad so the ocular signs such as discharge, conjunctival hyperemia or keratitis could be significant to make a definitive diagnosis (7, 9, 13, 18).

The Seidel test

One of the advantages of fluorescein is the fact that sterile solution is not harmful to intraocular tissues. It may be used to detect small corneal perforations, which are not readily visible without dye. To perform Seidel test ,fluorescein is applied as described above. If corneal perforation is present, aqueous humor leakage locally dilutes dye, which is visible as a dark area. This dark area is exciting wave of aqueous humor, which increase in size and flows downward over the cornea as the leak continues, pushing fluorescein away. It is easy to observe with magnification.

Fluorescein angiography

Fluorescein angiography (FA) was originally described by Navotny and Alvis in 1961 (23). It is a diagnostic imaging modality that provides direct visualization of retinal and choriocapillary vasculature. It is mainly used to determine the integrity of blood ocular barriers. Fluorescein angiography allows to evaluate posterior segment neoplasm, hypertension, retinal detachment, inflammatory processes, diabetic retinopathy and degenerative processes. FA in veterinary medicine is performed under deep sedation or anesthesia to avoid eye movement. The dose of fluorescein is 20mg/kg of 10% solution administrated intravenously in rapid bolus (23). Side effects such as emesis were reported in animals, nausea, vomiting and even fatal anaphylaxis in humans. In Kwan’s study from 2006 from total of 11898 FA adverse reactions were recorded in 132 cases, which gives 1,1% (15). After bolus injection, fundus photographs are performed using fundus camera. Camera is fitted with exciter and barrier interference filters, which, respectively, stimulate fluorescence by the dye and filter out other wavelengths of light reflected from the fundus. The process of fluorescein angiography is characterized by flow patterns of the choroidal and retinal vasculature, which is divided into a temporal phases (23). The first phase is a choroidal phase, In which the larger choroidal arteries and choriocappilaris are filled. The second one is an arterial phase, where retinal arteries, cappilaries and small retinal venules are completely filled. The venous phase is characterized by filling of the primary venules. The last, fourth phase is a recirculation phase and is associated with recirculation of fluorescein into retinal and choroidal vasculature. In healthy animals, fluorescein is excreted by the kidneys within 24 hours, resulting urine discoloration.

Fluorescein is a safe dye widely used in human and veterinary ophthalmology. It allows to perform a few diagnostic tests which are affordable/low cost, rapid and helpful to set further proceedings. Some disadvantages include wide ranges in tear breakup time or false results in case of Jones test, especially in brachycephalic breeds. They remain, nevertheless, first-line examinations. Not performing the Seidel test and thus not detecting perforation may have very serious consequences. Some ophthalmic preparations due to their chemical composition cannot get into the anterior chamber and they are toxic to the interior structures of the eye. Passing them will bring more losses than gains. The Seidel test is also recommended because of the size of corneal perforation, which may be inferior to 1 mm.

Fluorescein angiography is very widely used in human patients, and there is an abundance of medical articles on the subject. FA is limited in veterinary medicine because of the cost of the procedure, necessary equipment and differences of anatomy such as persistence of tapetum lucidum or degree of pigmentation. The method is also used in investigations of the anterior segment (iris lesions), but it is less informative with dark irises, which excludes most animals. It is important to keep in mind that fluorescent angiography is a new field in ophthalmology, the number of animal reports on this subject increasing. It is a future of human ophthalmology as well as veterinary medicine.


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