HOW-TO SESSION: CLINICAL PATHOLOGY


Slides should be packaged in a slide holder sur- rounded by bubble wrap or polystyrene packing mate- rial and submitted in a sturdy container to avoid breakage during transport. Simple cardboard mail- ers or thin padded envelopes, even if labeled “fragile,” “breakable,” or “glass” are not adequate to protect the slides. Alternatively, slides may be submitted indi- vidually in a pill container prior to protective pad- ding.4 Slides should be protected against moisture to avoid artifacts and never submitted in the same con- tainer as formalin-fixed tissue. Formalin fumes will fix the cells and render them unstainable.5 To collect hair for suspected dermatophytosis,


hairs should be plucked from the leading edge of a lesion. Alternatively, hair and crusts may be col- lected by brushing with a clean, small, stiff brush (such a toothbrush) or scraped with a scalpel at a lesion edge. Collected material should be placed in a test tube with a cotton plug or in a paper envelope. Moisture in a sealed tube may result in a prolifera- tion of saprophytic fungi.8 These samples may be used for both cytology and culture.


Microbiology


Bacteriologic culture and sensitivity testing is necessary when initial therapy does not work, in deep skin infections with draining tracts or gran- ulomas, when other bacteria besides cocci are seen on skin cytology, or if methicillin-resistant Staph- ylococcus is suspected.9 Any moist lesion will contain bacteria after 24 hours; therefore, attempt to culture lesions as soon as they occur or by opening a closed pustule. Samples may be ob- tained with a moistened cotton or nylon-tipped swab, by aspiration or from a tissue biopsy. Ster- ile equipment and aseptic technique should be used. Swabs or tissue samples should be ob- tained from the tissue exudate interface where the proliferation of organisms occur.9 Aspirated sam- ples of purulent exudate generally are sparsely populated or devoid of live organisms. Multiple swabs should be obtained from the lesion site given that the laboratory will often set up a num- ber of different tests on day 1 based on the infor- mation provided by the clinician.b Swabs should be immediately placed in a non-nutritive trans- port media (such as a commercial culturette, www.veterinarylabsupply.com/culturettes.htm), and kept cool, as microorganisms die rapidly in dry conditions. Whenever possible submit tissue rather than tissue swabs.b Tissue samples should be aseptically collected, wrapped in sterile gauze moistened with saline or lactated Ringer’s solu- tion (LRS) and placed in an airtight sterile con- tainer. A ring block or regional anesthesia is preferred when obtaining a biopsy for culture be- cause lidocaine and epinephrine inhibit the growth of many Gram-positive and Gram-negative bacteria, mycobacteria, and fungi.2 An unstained air dried and or fixed slide for a Gram stain should accompany the samples for microbial propagation.


Samples should be transported to the laboratory as rapidly as possible; ice packs and overnight shipping are recommended.c Knowledge of the lab- oratory schedule is helpful if one can plan sample collection. Samples should arrive, if possible, in the morning so that tests may be set up on the arrival date. Ideally the diagnostic laboratory would operate fully 7 days a week and on holidays; however, many laboratories have only one, or a few, personnel that set up tests received on Friday afternoon or on Saturday, and subsequent tests are often delayed until the following Monday.b Limitations of bacterial culture include an over-


growth of contaminating organisms, organisms that do not grow or are slow growing, deficient culture technique, delayed shipment of the sample, and con- current antibiotic therapy at the time of sampling. Organisms that are hard to grow may be identified by molecular techniques from tissue samples.10,11 If any unusual results occur, or if no growth occurs, consultation with the diagnostic laboratory microbi- ologist is advisable. Samples submitted for culture of dermatophytes


should be collected as described above. If a deep fungal infection is suspected a tissue sample is best for culture and molecular diagnostic techniques. A sample of tissue for histopathological evaluation should also be submitted to confirm that fungal iso- lates are pathogenic.11


Biopsy


“When the clinician and pathologist truly work to- gether, the skin biopsy can reflect the dermatologic diagnosis in more than 90% of cases.”2 Yet many clinicians are frustrated with vague diagnoses or descriptions of biopsies that leave the clinician and owner with the impression of little useful informa- tion. The selection of biopsy site, procurement of the biopsy, and proper handling and submission of biopsies are crucial to an informative interpretation of submitted material. A skin biopsy should be taken of any unusual or significant lesions, lesions that do not respond to empirical therapy in 3 weeks, any persistent ulcerated lesion, all neoplastic or sus- pected neoplastic diseases, when the biopsy is likely to be the definitive diagnostic test (such as immune- mediated diseases), and any lesions(s) where the treatment is likely to be time consuming, expensive, or dangerous.12 Biopsies are best obtained from early developing


lesions and not chronic ulcerated lesions. This step may require a careful complete examination of skin, especially if there is a generalized distribution of lesions. Biopsies from excoriated or traumatized lesions, found in many pruritic conditions, are not likely to be informative. Anti-inflammatory medi- cations, especially corticosteroids, will alter an in- flammatory disease process, rendering a biopsy less informative. Most authors recommend discontinu- ation of oral corticosteroids 2–3 weeks prior to a biopsy and 4–6 weeks if long-acting corticosteroids


AAEP PROCEEDINGS  Vol. 61  2015 149


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