22 May 2020
Kimberley Davies, BVSc, MRCVS and Fernando Malalana DVM, GPCert(EqP), DipECEIM, MRCVS explain how to obtain and interpret samples in the field, to help with first opinion practitioner competency.
Corneal disease is commonly encountered in equine first opinion practice, with corneal ulceration making up a significant proportion of this population.
The equine cornea is less than 1mm thick and can demonstrate a poor ability to heal (Herbig and Eule, 2015); therefore, corneal disease can rapidly deteriorate if prompt and appropriate treatment is not initiated as early on in the disease process as possible. Many testing modalities are routinely used to help the practitioner assess corneal disease, including ophthalmoscopy, fluorescein staining and culture; however, cytology is often not performed.
Corneal cytology is a simple and economic technique enabling a rapid diagnosis to be made and appropriate therapy to be initiated in some cases. This article discusses how to obtain and interpret samples in the field, enabling all first opinion practitioners to become competent at corneal cytology.
Cytology can be performed at any stage during the disease process; however, like many diseases, the sooner a diagnosis is made, the sooner appropriate therapy can be initiated, thereby reducing the complication rate. Cytology should be performed in all cases of corneal ulceration and become part of the practitioners’ usual routine.
All practitioners should routinely carry equipment in the car to be able to perform cytology, and every lab should be equipped with Diff-Quik stain, Gram stain and a microscope. Stains should be kept specifically for ocular cases to prevent cross-contamination and subsequent false results.
Limitations to corneal cytology include non-exfoliative lesions where a sample cannot be obtained. Results occasionally require careful interpretation, as a negative sample may not necessarily mean a negative result if the clinician suspects a particular disease – for example, in cases of mycotic keratitis.
Contraindications for performing corneal cytology include descemetocoeles and corneal perforations (Clode, 2012). These cases should be referred immediately to an ophthalmic specialist.
A clinical examination of the horse should routinely be performed, as well as a more focused examination of both eyes. The practitioner should use an adequate light source and undertake the examination in a quiet area. Cranial nerve examination should be undertaken, as well as a thorough examination of the eyelids, conjunctiva and nictitating membrane.
Once this part of the examination has been completed, the patient can be sedated and an auriculopalpebral nerve block placed (Hartley, 2014a). If culture is to be performed in addition to cytology, it should be done at this stage prior to any contamination of the sample site (Gilger, 2017). Topical local anaesthetic should then be placed into the affected eye.
Samples can be taken using a cytobrush (this is the best option as superior cell preservation exists compared to other techniques), a microbiology swab, the blunt end of a scalpel blade or a kimura spatula (Clode, 2012).
Techniques are:
Cytobrush/microbiology swab technique – roll lightly over the margins of the ulcer. Roll the brush on to two to four microscope slides.
Scalpel/Kimura spatula technique – hold at 45° to the corneal surface and gently scrape the ulcer margins. Spread a thin layer of the sample on to two to four microscope slides.
If no sample can be visualised on the slides then repeat the technique. Slides should be appropriately labelled with the horse and owner’s name, date and the eye that was sampled. Place the slides into a slide holder to air dry during transportation back to the practice.
Once back in the practice, one slide should be stained using Diff-Quik stain, gently rinsed with water and air dried. The slide should then be examined under the microscope.
If evidence of bacteria exists then a second slide should be prepared using Gram stain. If fungal keratitis is suspected then a request for periodic acid-Schiff stain at an external lab can be made (Cowell and Tyler, 2002).
Cytological evaluation of a normal cornea will reveal sheets of epithelial cells. Staining with Diff-Quik highlights superficial epithelial cells as flat structures containing large amounts of blue cytoplasm and basophilic nuclei.
Epithelial cells from the intermediate cornea appear polyhedral with basal layer cells appearing cylindrical in shape and which stain more darkly due to a reduction in cytoplasm (Gilger, 2017). Assessing the type of cell present enables the practitioner to determine the depth of the lesion.
Care must be taken over individual cells that may appear rolled up at the edge of the sample as they can occasionally be mistaken for foreign bodies or branching fungal hyphae. Degenerate corneal material may be seen as “cytoplasmic streaming” and can be easily mistaken for fungal hyphae (Gilger, 2017).
One of the most common findings observed on cytology is the infiltration of neutrophils that may have toxic changes. Occasionally, lymphocytes, monocytes and plasma cells may be seen.
Although a suppurative infiltrate is not pathognomonic for infection, it gives the practitioner reason to be highly suspicious. To confirm the presence of bacterial infection, a Gram stain should be performed. It is highly likely bacteria will be present, with intracellular bacteria confirming the presence of bacterial keratitis that requires treatment.
The most common bacteria found in ulcerative keratitis include Streptococcus, Staphylococcus and Pseudomonas species (Gilger, 2017). Gram staining will enable identification of the inciting pathogen and an appropriate treatment protocol can be formulated while awaiting culture results.
Eosinophils can be identified on cytological examination of horses with idiopathic eosinophilic keratitis. In addition, sparse mast cells, neutrophils, lymphocytes and plasma cells may be seen.
Fungal infections were previously deemed to be rare in the UK; however, they are increasing in prevalence, lending even more importance to the use of routine cytology in ulcerative keratitis cases (Hartley, 2014b).
Fungal hyphae stain well with Diff-Quik stain and appear as a branching structure with parallel walls where occasional budding spores can be seen.
The presence of even a few fungal hyphae is pathognomonic for mycotic keratitis; however, if no hyphae are seen, but mycotic keratitis is suspected based on clinical examination, repeat sampling should be performed. It may be the fungal hyphae are deep to the superficial cornea – for example, in a stromal abscess.
The mineralised deposits stain light blue with Diff-Quik stain and correlate with calcium deposits in subepithelial layers of cornea from horses with stromal keratopathies (Gilger, 2017).
Occasionally, red blood cells can be identified if the disease process has involved haemorrhage.
Corneal cytology is an invaluable adjunctive tool to any equine practitioner desiring rapid diagnoses to corneal disease. It should be used in conjunction with a thorough clinical examination and alongside other testing modalities – including culture – to determine an accurate diagnosis and development of an appropriate treatment strategy.
With its limitations borne in mind, corneal cytology is a simple, inexpensive testing modality requiring minimal training to master, which can provide a rapid diagnosis and faster initiation of appropriate treatment protocols, thereby reducing healing times and improving client satisfaction.
