Laboratory investigation

2 Laboratory investigation


The collection and handling of samples from the eyes is commonly performed in veterinary ophthalmology. Samples collected include swabs, scrapings and other samples for cytology, aspirates and biopsy specimens. They are useful for the investigation of suspected infectious disease, as well as immune-mediated and neoplastic processes.


Swabs can be taken from the conjunctiva, cornea or discharges. It is important to tell the laboratory exactly where the sample has been taken from. A sterile swab is gently rolled across the appropriate surface and topical anaesthesia is not usually required (it can affect interpretation of the results and can have a bacteriostatic effect which is not desirable when trying to isolate such pathogens!). If the sample is for cytology, then a dry swab is used and cellular integrity is usually preserved; however, there are better methods of sample collection for cytology, as discussed below.


The majority of swabs taken are for microbial analysis, and in this instance it is better to use a damp swab (moistened either with sterile saline or the appropriate culture medium). This gives a better yield of pathogens. It is important not to touch the swab against other parts of the eye since it could become contaminated with bacteria which are normally present on the ocular surface. Swabs should be labelled and dated prior to being dispatched. Previous and current medication should be detailed since this can affect interpretation of the results. If there is any delay between sample collection and processing, then the swab should be refrigerated (but not frozen). The culture requirements for bacteria, chlamydophila, mycoplasma, fungi and viruses can all vary and if there is any uncertainty about the best medium for collection and so on, then it is important to discuss this with the laboratory. In addition to culture, the sample should undergo susceptibility testing for aerobic bacteria.


Swabs taken for viral isolation or polymerase chain reaction (PCR) are usually for feline herpes virus (FHV-1) and special culture media are required. Combined media for FHV-1 and chlamydophila are routinely used. Swabs are usually obtained from both the conjunctiva and the oropharynx. These samples can be frozen if there is any delay in dispatch to the laboratory (over the weekend for example) but it is always sensible to check this with the laboratory first.


Scrapings are usually used for cytological examination. They are useful for inflammatory and neoplastic conditions mainly, but are of use in infectious processes as well. Samples can be taken from eyelids in exactly the same way as from other skin sites – a scalpel blade is used and the tissue gently squeezed during the scraping to express material from the glands and hair follicles. It is important that the eyelid margins are not damaged, and since the procedure can result in some swelling and discomfort afterwards, the animal must be monitored to prevent self-trauma. Scrapings from the conjunctiva and cornea require topical anaesthesia, and sometimes sedation of the patient. Holding a sterile cotton bud moistened with topical anaesthesia against the area to be sampled for 30–60 seconds will achieve a deeper numbing. A specialized instrument is available for scraping the cornea and conjunctiva – the Kimura spatula. However, most people use the blunt end of a scalpel blade for convenience. The material obtained is gently spread across a dry microscope slide.


Another way of collecting samples for cytology is to use a cytobrush. These are small nylon-bristled brushes that look like mascara-wands. They were initially designed for gynaecological use but are popular for ocular cytology since they are safe to use and maintain good cellular integrity as well as distributing the cells evenly. Once the material is collected by gently rolling the brush over the surface of the tissue, it is then rolled again over a dry microscope slide.


A final technique for sample collection for cytology is the impression smear. This is again very easy to perform – a dry microscope slide is pressed firmly against the tissue to be sampled – any cells which readily exfoliate are transferred to the slide.


Whichever method of sample collection is used, the principles for processing the specimens are the same. Samples are gently spread on a clean microscope slide and air dried. In-house assessment using Diff-quik stains and Gram stains can be useful for bacterial identification, the presence of fungi and evaluation of cell types, but samples should also be sent to a cytologist for professional interpretation; it is useful to send unstained samples as well since further specialist stains can be used if indicated – for example, immunofluorescence stains for feline herpes virus and chlamydophila.


Aspirates can be taken from ocular tissue in the same way as sampling from elsewhere in the body. They are useful for investigating masses involving the adnexa, orbit or the eye itself. Abscesses, granulomas, tumours such as lymphomas, mast cell tumours and cysts can be differentiated. Inadvertent globe damage can be avoided by making sure that the animal is suitably restrained (with light sedation if necessary) and always directing the needle away from the globe. The area to be sampled is stabilized if possible, and the needle is inserted without the syringe attached. Suction is then applied, and the needle is redirected several times to get a representative sample. The syringe is removed, filled with air, and then reconnected to the needle. The sample obtained is then expressed onto a microscope slide. A second slide is used to gently spread the material. Occasionally it is necessary to centrifuge the sample prior to smear preparation if cellular yield is likely to be low (e.g. samples taken from the anterior chamber). In addition to cytology, microbial analysis can be performed on aspirates by placing the material on a moistened swab as detailed above.


Biopsy samples can be taken where the pathological process is too deep for scrapes and smears to be of use, or when the actual architecture of the tissues, rather than just the cells involved, needs to be examined. Samples should be placed in their correct orientation on card before putting in the fixative to make interpretation easier. If necessary, samples can be sent for microbial investigation as well as histology. Conjunctival biopsies are taken after the area has been desensitized with a cotton bud soaked in topical anaesthetic as mentioned above. An area of conjunctiva is tented up using a fine pair of rat-toothed forceps, and a piece is snipped off from below using small sharp scissors. No sutures are needed as the area heals rapidly. Some haemorrhage can be expected but this soon stops if gentle pressure is applied.


Eyelid biopsies are taken under general anaesthesia if wedge sections including the eyelid margin are needed. Sometimes punch biopsies can be taken with the animal sedated. Histology can be performed on biopsy samples from the cornea – these are obtained by surgical keratectomy under general anaesthesia.


A complete histological examination is frequently performed on enucleated globes. Examples include painful blind eyes due to refractive uveitis or hyphaema, glaucoma or endophthalmitis. Histology can help to understand the pathological processes which lead to loss of the eye and can be useful for ongoing case management – typing tumours for example. Correct handling of the specimen is required prior to submission to the laboratory. All tissue apart from the optic nerve should be trimmed from the globe – the histopathologist does not require eyelids and muscle if the pathology is contained within the eye. If there are concerns about the periocular tissues (e.g. tumour spread), then these can be submitted separately if necessary – leaving them on the enucleated globe will only reduce penetration of the fixative. Most globes will be fixed in formalin, but several specialist media are available and consultation with the pathologist might be useful.


All fixatives are potentially dangerous and so due attention to health and safety issues must be paid if handling them. It is important to give the pathologist as much information as possible – in addition to the animal’s details and which eye is submitted, information about the clinician’s findings should be included – a copy of their ophthalmic examination report together with diagrams of any focal lesions will be of great assistance. Details of any medical or surgical treatments plus any findings in the fellow eye should be submitted.


Related posts:

  1. Blepharitis
  2. Proliferative (eosinophilic) keratoconjunctivitis
  3. Limbal melanoma
  4. Canine lens luxation

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Sep 10, 2016 | Posted by in SMALL ANIMAL | Comments Off on Laboratory investigation

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