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Fluorescein
by Noelle La Croix, DVM, Dip. ACVO

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Fluorescein is a fluorescent dye first synthesized by the Nobel prize-winning chemist Albert Baeyer in 1871.  It is derived from aromatic compounds (benzene and its derivatives) and contains four aromatic rings of carbon (Figure 1).  In these aromatic compounds delocalized (resonating) electrons form doughnut-shaped rings that hover above and below the plane of carbon nuclei.  This resonance is responsible for the color and the fluorescent properties of fluorescein.

Bonded electrons within compounds can be excited to higher quantum states by absorbing energy (such as photons of light).  Higher energy states are generally unstable and will resolve back to ground states by releasing the absorbed energy.  This released energy can be in the form of light as well.  Compounds which absorb light energy and then release it as photons of light are said to be fluorescent.  The fluorescein molecule's resonating electrons maximally absorb blue light (465 to 490 nm) energy and then emit lower energy (higher wavelength) photons of yellow-green light (520 to 530 nm).  Hence, veterinary ophthalmologists best view fluorescein with the blue light of a slit-lamp.

In ophthalmic examinations fluorescein is routinely used to detect physical breaks in the corneal epithelium.  The dye does not actually bond or stain tissues, but it is easily visualized even when greatly diluted.  In high concentrations fluorescein appears orange, but in dilute concentrations green.  An intact corneal epithelium has a high lipid content that resists the penetration of fluorescein and so is not colored by it.  A break in the corneal epithelium allows water-soluble fluorescein to be absorbed by the hydrophilic corneal stroma.  The exposed, and now stained, corneal stroma will therefore fluoresce green.  If large areas of the cornea are denuded (i.e., 80 to 90%) then applied fluorescein can even penetrate into the aqueous humour.

Another common use for fluorescein is the Siedel test which detects aqueous humour leakage from a deep corneal wound (ie; a leaking corneal ulcer).  In the test, a fluorescein-impregnated paper strip makes contact with an area of possible corneal rupture and leakage.  The strip's high concentration of fluorescein give it an orange appearance.  The eyelids are then held open and viewed under blue light.  A trickle of green fluorescent fluid emanating from the strip-contacted area indicates a leakage of aqueous humor through a corneal rupture.  Active corneal ruptures require immediate surgical repair.

The Jones test also relies upon fluorescein to determine nasolacrimal duct patency.  In this test, fluorescein is first placed within a conjunctival sac.  If the dye is later detected at the ipsilateral naris the nasolacrimal duct is patent.  The test can be timed and performed with either a commercially-available fluorescein solution or a fluorescein-impregnated strip (Figure 2).  Normal dogs tested with a fluorescein-impregnated strip have an average nasolacrimal transit time of 248 seconds.  However, dogs tested with a fluorescein solution have an average nasolacrimal transit time of only 48 seconds.  In addition, brachycephalic dogs generally have prolonged nasolacrimal fluorescein transit times.  Normal cats tested with a fluorescein-impregnated strip have an average nasolacrimal transit time of 30 seconds.  However, some normal cats do not passage fluorescein-impregnated strip stain to the nares at all.  All normal cats tested with a fluorescein solution will show passage of the dye within 30 minutes.

An advanced fluorescein-based technique that is sometimes performed by a veterinary ophthalmologist is fluorescein angiography.  It involves a systemic injection of fluorescein followed by observation of the dye as it circulates through the choroid, retinal arterioles, capillaries, and venules.  A high speed digital camera is used to photograph different phases of retinal circulation in an anesthetized veterinary patient.  Fluorescein angiography is useful in diagnosing retinal pathology.  For example, the technique can be used to determine if a retinal lesion is a tumor or a blood clot: a tumor's blood vasculature will fluoresce, but a clot will not.

Corneal staining, the Siedel test, and the Jones test can all be routinely performed in your practice during a veterinary examination of the eye.  The fluorescein used in these tests will reveal corneal ulceration, aqueous humour leakage, and problems with nasolacrimal duct patency.  Fluorescein angiography performed by an ophthalmologist can also be used to diagnose retinal lesions.  If you have any questions or concerns about fluorescein please feel free to contact a veterinary ophthalmologist.

Noelle La Croix, DVM, Dip. ACVO
Veterinary Medical Center of Long Island
75 Sunrise Highway
West Islip, New York 11795
(631) 587-0800; fax (631) 587-2006

http://www.vmcli.com

Figure 1: Fluorescein is a fluorescent dye first synthesized by the Nobel prize-winning chemist Albert Baeyer in 1871. It is derived from aromatic compounds (benzene and its derivatives) and contains four aromatic rings of carbon.

Figure 2: The Jones test also relies upon fluorescein to determine nasolacrimal duct patency. In this test, fluorescein is first placed within a conjunctival sac. If the dye is later detected at the ipsilateral naris the nasolacrimal duct is patent. The test can be timed and performed with either a commercially-available fluorescein solution or a fluorescein-impregnated strip.

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