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					                                  80th Western Veterinary Conference



V231
Otic Cytology
Lynette K. Cole, DVM, MS, DACVD
The Ohio State University, College of Veterinary Medicine
Columbus, OH, USA

OBJECTIVES OF THE PRESENTATION
    To become familiar with performing and evaluating otic cytology.

GENERAL KEY POINTS
    Otic cytology is quick and easy to perform.
    Otic cytology is an important diagnostic procedure used for determining what, if any, infective
    organisms are present in the ear.
    Mean counts of yeast and bacterial organisms per high-power dry field (400x) from otic swabs in
    the dog of ≥ 5 and ≥ 25, respectively, are considered abnormally increased.
    Mean counts of yeast and bacterial organisms per high-power dry field (400x) from otic swabs in
    the cat of ≥ 12 and ≥ 15, respectively, are considered abnormally increased.
    Artifacts may be present cytologically and be mistaken for infectious organisms, such artifact is
    melanin granules and stain precipitates.

OVERVIEW OF THE ISSUE
Otic cytology is an important diagnostic procedure used for determining what, if any, infective organisms
are present in the ear, as well as the types of inflammatory cells, in animals presented with the complaint
of otitis externa.
Utility of Cytology
Unfortunately, there is no reliable correlation between the nature of otic discharge and a specific
organism. In some instances, the exudate is only wax. Therefore, it is important to sample the exudate in
the ear for cytologic evaluation in all animals suspected of otitis externa. The technique is quick and
simple, and allows one to begin treatment immediately based on the results obtained. It is important to
perform cytologic evaluation of otic exudate at the initial examination as well as each recheck, in order to
monitor the response to the therapy. In addition, cytology is the best diagnostic technique for
identification of yeast organisms.
Sampling Collection and Preparation
After the ears have been examined otoscopically, exudate from the horizontal ear canal is collected by
inserting a cotton-tipped applicator into the canal. Samples should be collected from both ear canals. The
swab samples are rolled onto a glass microscope slide. If a frosted edge slide is used, roll the sample from
the left ear next to the frosted edge, and the sample from the right ear on the opposite side of the slide. By
doing this, only one slide is required for both ears. The next step is to heat fix the slide. Performing this
step will prevent a waxy sample from being washed away during staining. The sample is now ready to
be stained. The most commonly used stains are Romanowsky-type stains (Wright’s stain, Giemsa stain,
Diff-Quik), since they are very rapid and easy to perform, compared to the more cumbersome and time-
consuming Gram stain. The disadvantage of these stains is that all bacteria stain blue and the gram-
staining properties cannot be determined. After staining, the slides should be rinsed with cool water and
allowed to dry. Drying may be expedited with the use of a hand-held dryer.
Viewing and Evaluation
Once the slide is dry, the cytologic smear should be viewed beginning with a low power magnification
and then advancing to a higher magnification. The slide is first scanned with the low power lens (10x
objective; 100x magnification) to find keratinized squamous epithelial cells or inflammatory cells. This is
where infective organisms would be if they are present. Once these are located, I will go directly to oil
immersion (100x objective; 1000x magnification) to evaluate the cells and any infective organisms. The
smears should be evaluated for the number and morphology of bacteria, the number of yeast, the number
and type of leukocyte, the presence of excessive cerumen, the presence of excessive keratinized squamous
epithelial cells, and the presence of neoplastic cells. I will routinely evaluate 10 fields under oil immersion
and estimate the number of organisms and leukocytes per those 10 fields. If the mean number of bacterial
or yeast organisms is ≥ 4, then the organisms should be considered pathogens. For consistency, a grading
system of the numbers of organisms and leukocytes counted should be developed.
Cytologic Appearance of the Normal Ear Canal Compared to the Otitic Ear
Cerumen coats the external ear canal and is composed of secretions from the sebaceous glands,
ceruminous glands as well as sloughed keratinized squamous epithelial cells. Cerumen is clear and
usually does not stain due to its high lipid content. Ears that are inflamed will have a lower lipid content
than normal ears resulting cytologically in a stained smear that is bluer than a smear from a normal ear.
Keratinized squamous epithelial cells are usually anucleate and stain blue/purple.
       A recent study evaluated numbers of inflammatory cells, epithelial cells, bacteria and yeast
organisms cytologically from ear swab samples from normal dogs and cats and compared these to the
results obtained from dogs and cats with otitis externa. The slides were scanned at low power (100x) to
locate inflammatory cells or keratinized squamous epithelial cells where infectious organisms were
expected to be found. Ten fields were counted under high-dry power field (400x) and the mean number
of cells and/or organisms per sample was calculated. Inflammatory cells were absent in all the samples
from the normal ears. Inflammatory cells were abundant in affected ears with purulent otitis but were
absent in 36% of the samples obtained from affected ears with ceruminous exudate. It is interesting to
note that the authors did not find any significant difference in the mean numbers of keratinized
squamous epithelial cells between the normal and affected ears. It is believed that in cases of chronic otitis
externa and diseases with increased epithelial turnover rate (keratinization disorders) or atopic
dermatitis, there would be increased numbers of keratinized squamous epithelial cells. One explanation is
that the selection of the fields for counting was based on finding keratinized squamous epithelial cells,
which may have resulted in a higher density of these cells from the dogs with normal ears.
       The authors next evaluated yeast and bacterial organisms from the swab samples of the normal and
affected dogs and cats. Malassezia pachydermatis is identified morphologically as a broad-based, budding
yeast, typically “peanut-shaped”. Bacterial organisms are identified morphologically as cocci and rods.
Ten selected fields were counted under high-dry power field (400x) and the mean number of organisms
per sample was calculated. In dogs and cats with clinical signs of otitis externa, the mean number of
Malassezia organisms was significantly higher than from the dogs and cats with normal ears. Only coccoid
bacteria were identified in swab samples from normal ears. In the dogs and cats with clinical signs of
otitis externa, the mean counts of bacteria were significantly higher than from the dogs and cats with
normal ears. According to the results of this study, mean counts of yeast and bacterial organisms per
high-power dry field (400x) from otic swabs in the dog of ≥ 5 and ≥ 25, respectively, are considered
abnormally increased, while mean counts of yeast and bacterial organisms per high-power dry field
(400x) from otic swabs in the cat of ≥ 12 and ≥ 15, respectively, are considered abnormally increased.
       It would be unusual to find cytologic evidence of neoplasia in the otic exudate from an ear with a
mass. Cytology would be used to identify any secondary infectious organisms or inflammatory cells.
Cytologic Appearance of Artifacts
Artifacts may be present cytologically and be mistaken for infectious organisms. One such artifact is
melanin granules. Melanin granules may be present in the keratinized squamous epithelial cells and
should not be confused with rod bacteria. The melanin granules will stain brown, while rod bacteria will
stain blue/black.
       Precipitates on the slide stain blue/black and may be mistaken for coccoid bacteria. They differ
from bacteria, since they form large, indistinguishable clumps while coccoid bacteria are very round and
uniform in size. This artifact is due to inadequate stain filtration. This can be avoided by periodically
filtering the stain and replenishing the buffer solution.
       Treatment should begin based on the results obtained cytologically. If there is only cerumen and
keratinized squamous epithelial cells present, an ear cleaning solution should be dispensed. If infectious
organisms are identified, treatment for the specific organism(s) should be started. If rod bacteria are
found cytologically, a culture and susceptibility should be performed to determine the type of bacteria
present. The most common rod bacteria from chronically infected ears is Pseudomonas aeruginosa which
can be difficult to treat, since it may be resistant to numerous antibiotics. While awaiting culture results,
assume the infection to be P. aeruginosa, and begin treatment accordingly. In addition, primary
underlying diseases such as primary idiopathic seborrhea, atopic dermatitis, food allergy,
endocrinopathies, and Otodectes should be considered in cases of chronic, recurrent otitis externa.

SUMMARY
Treatment should begin based on the results obtained cytologically. If there is only cerumen and
keratinized squamous epithelial cells present, an ear cleaning solution should be dispensed. If infectious
organisms are identified, treatment for the specific organism(s) should be started. If rod bacteria are
found cytologically, a culture and susceptibility should be performed to determine the type of bacteria
present. The most common rod bacteria from chronically infected ears is Pseudomonas aeruginosa which
can be difficult to treat, since it may be resistant to numerous antibiotics. While awaiting culture results,
assume the infection to be P. aeruginosa, and begin treatment accordingly. In addition, primary
underlying diseases such as primary idiopathic seborrhea, atopic dermatitis, food allergy,
endocrinopathies, and Otodectes should be considered in cases of chronic, recurrent otitis externa.

				
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posted:1/21/2011
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