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Laboratory investigation of ophthalmic disease

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Abstract

Laboratory investigation is an important diagnostic tool in veterinary ophthalmology. As well as being the primary target for many diseases, the eye is also often involved in systemic disease processes, including infectious, neoplastic and immune-mediated conditions. Laboratory investigation can be the gateway to the accurate diagnosis of such diseases, which in turn plays a fundamental role in patient care and management. This chapter considers sample collection and handling; microbiological investigation; cytological evaluation; histopathological examination.

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Figures

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3.2 Technique for preparing a cytology smear. Note that the slides can also be held perpendicular to each other, rather than parallel, before smearing. A scrape of material from a region of chronic superficial keratitis (pannus) in a German Shepherd Dog, which has been correctly smeared. A mixture of non-degenerate neutrophils and the occasional lymphocyte can be seen. (Wright–Giemsa stain; original magnification x1000). Drawn by S.J. Elmhurst BA Hons (www.livingart.org.uk) and reproduced with her permission.
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3.3 Impression smear of a third eyelid lesion showing a mixture of anucleated keratinized squames, keratin scrolls and a low number of granulocytes. (Wright–Giemsa stain; original magnification x100) Fine-needle aspirate of the same lesion. A background of red blood cells can be seen, through which is scattered a moderate number of variably granulated mast cells. (Wright–Giemsa stain; original magnification X400)
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3.4 Aqueous humour collected 10 days following phacoemulsification from a dog with endophthalmitis. Cellularity is increased and the population is mixed with non-degenerate neutrophils and mononuclear cells evident. (Wright–Giemsa stain; original magnification x100) Magnified view. A mixed population of non-degenerate neutrophils, macrophages and the occasional lymphocyte is visible. The macrophages contain a dark green–black pigment, which may be melanin. The granular material throughout the background is stain, not bacteria. (Wright–Giemsa stain; original magnification x400)
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3.5 A collection of epithelial cells and eosinophils from a patient with eosinophilic keratitis. The large pool of blue–purple granular material is stain deposit and should not be confused with bacteria. (Wright–Giemsa stain; original magnification x400)
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3.6 A small group of epithelial cells surrounded by lubricating gel. The lubricating gel appears as dark purple amorphous granular material. (Wright–Giemsa stain; original magnification x400)
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3.7 A biopsy capsule can be useful, particularly for small specimens. The biopsy capsule containing the specimen is then placed in a formalin-filled container.
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3.8 Non-diseased extraocular tissue should be trimmed off the globe prior to immersion in fixative. The dotted line marks the approximate position of the globe. The extraocular tissue has not been removed. This is a common error and results in the tissue:formalin ratio being too large. This globe has been properly prepared and fixed. The eyelids, extraocular muscles and fat have been removed and the attached optic nerve has been left intact.
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3.9 An exenterated specimen of a canine orbital meningioma. This tumour is best left attached to the globe as it will help with orientation and assessment of surgical margins. Adenocarcinoma of the third eyelid gland in a dog. This tissue should not be trimmed off the globe. Conjunctival melanoma in a cat. This tissue should not be trimmed off the globe.
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3.10 A globe from a dog with anterior scleritis that has been fixed with Davidson’s solution. Note the generalized white opacification of the tissue, which can make macroscopic examination and photography difficult.
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3.11 The submitted globe is macroscopically examined and then sectioned adjacent to the optic nerve to reveal an extensive pigmented mass that proved to be a choroidal melanoma. Half the globe is then placed in a cassette. The cassette undergoes routine processing, which involves dehydration of the tissue, permeation with a clearing agent (solvent) and then replacement of the solvent with a wax. The processed part of the globe is embedded in a firm wax block from which 4 μm sections can be cut. The 4 μm section is mounted on a glass slide and routinely stained with H&E. Histopathological examination reveals neoplastic melanocytes. (H&E stain; original magnification x400)
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3.12 GMS stain highlights fungal hyphae, which stain black. (Original magnification x400) ZN stain confirms the presence of acid–fast organisms, which stain bright red (typical of spp.) (Original magnification x1000)
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3.13 Macroscopic examination of a feline globe showing a solid white intraocular mass, which partially replaces the uveal tract. Histopathological examination reveals sheets of large neoplastic lymphocytes, indicative of lymphoma. (H&E stain; original magnification x400) Immunohistochemistry indicates that this is a T-cell lymphoma. The neoplastic lymphocytes are immunopositive for the T-cell marker CD3. Immunopositivity is characterized by the brown staining. (Diaminobenzidine chromogen stain; original magnification x200)
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3.14 A feline globe with extensive proteinaceous exudation in the anterior chamber and vitreous, highly suspicious for feline infectious peritonitis (FIP). The dislocated lens is artefactual. Immunohistochemistry confirms the presence of feline coronavirus (FCoV) antigen within the macrophages, which is demonstrated by the brown immunopositive staining. (Diaminobenzidine chromogen stain; original magnification x200)

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