Microscopic examination of cytologic cutaneous preparations is a very useful, rapid, inexpensive test which may give us important information about the sampled lesions in less than 5 minutes. The necessary equipment is represented by slides with frosted ends, 5 ml syringes with 21 gauge needles, 24 gauge needles, swabs, acetate tape, staining liquids and a microscope with oil immersion objective is necessary for the observation of the specimens.
Sampling Techniques, Fixation and Staining
Preference should be given to the quality and not to the quantity of the cells collected, so that a thin, single cell layer should be obtained with smears.
Impression smears are useful in open exudating lesions, greasy seborrhoeic surfaces, and from freshly cut surfaces of skin biopsies or extirpated nodules. With this technique the glass slide is simply pressed repeatedly (not streaked) on the lesion. In a similar way pus from pustules and under crusts can be collected, after gently opening the pustules or the crusts with a small 25 gauge needle.
Skin scrapings performed with a n. 10 or 20 blade on greasy seborrhoeic material may be smeared on a glass slide, in order to look for Malassezia or very superficial bacteria.
Material for cytology can be collected with a swab from fistulas, deep pyoderma, ears or, with a moistened swab, from a greasy skin surface. The swab is then gently rolled across the slide.
Acetate tape preparations are performed by repeatedly pressing a strip of clear acetate tape on the skin, particularly on greasy areas. It is a suitable technique for the collection of seborrhoeic material for the search for Malassezia on the keratin scales. Acetate tapes can be stained as glass slides.
All cytological samples have to dry on the slide. Usual rapid stains used in cytology include modified Wright stains (Diff Quick, Hemacolor). Samples are immersed 5–10 seconds each in ethanol (fixation), in the red stain and in the blue stain, then shortly rinsed under tap water and air-dried. Acetate tape preparations can also be stained, rinsed and pressed to a microscope slide with the sticky face on the slide.
Sample Examination and Conservation
Slides are observed under the microscope at 4x, 10x, 40x and 100x (with oil immersion). The richness of the sample, the quality of the smear and the cellularity should be evaluated first at low power (4x). Groups of cells are identified at 10x, and these cells are then better observed at 40x and 100x. Only intact cells should be examined and evaluated. In order to keep the preparations for a longer period, a coverslip can be glued on the glass slide with special glues (e.g., Eukitt).
In case of inflammation several inflammatory cells are attracted to the lesions. The first ones to be attracted are neutrophils, particularly in case of skin infections caused by pyogenic bacteria. Young neutrophils have 3 nuclear segments, old neutrophils up to 5. These cells are the main phagocytic cells and are often observed with bacteria inside their cytoplasm. Neutrophils can degenerate because of age or bacterial toxins. In the first case the nucleus breaks down in several shrunk pieces (pyknosis, karyorrhexis), in the second case it swells excessively. Neutrophils are observed in all acute, subacute and chronic active inflammations, included immune-mediated diseases.
After some hours histiocytes (macrophages) may arrive. These cells are larger and have a kidney-shaped to round nucleus. In their cytoplasm, vacuoli may be observed, depending on what they have ingested. Macrophages are able to form multinucleated giant cells, often seen in foreign body reactions.
In chronic inflammations (e.g., after 7–10 days) lymphocytes and plasma cells may be observed. These cells have a small round nucleus. Plasma cells have a darker and larger cytoplasm than lymphocytes, with a visible clear spot, the Golgi apparatus. Plasma cells are responsible for antibody production, and when very active may contain Russel bodies.
In some occasions eosinophils may be observed. These granulocytes usually have a bilobated nuclei and contain large red granules. Mast cells are often seen associated to eosinophils. Mast cells are large round cells with a round nuclei, and contain large amounts of small violet (metachromatic) granules.
In an inflammatory exudate one can often observe the presence of bacteria, and evaluate their number, shape and position (intra- versus extracellular). Bacteria seen in cytology usually are cocci (mostly Staphylococcus), rarely rods or mixed infections (cocci and rods) can be observed. Because rods cannot be identified by cytology alone, finding intracellular rods or mixed infections warrants a bacterial culture and sensitivity test. Intracellular bacteria are diagnostic of pyoderma. In deep pyoderma usually few intracellular bacteria are observed, together with many neutrophils, macrophages, some lymphocytes and plasma cells. Numerous extracellular bacteria are indicative of contamination or of a very superficial pyoderma (such as a "hot spot").
Malassezia yeasts are rarely seen in samples from normal skin, can be normally present in healthy ears, and can be abundant in specimen from greasy seborrhea or ceruminous otitis. They are larger than most bacteria, have a bottle- or peanut shape (budding yeast), stain variably from light blue to dark violet and are always extracellular and often sticks to corneocytes. Other microorganisms, such as agents of deep fungal infections or Leishmania can be occasionally observed in samples from skin or lymph nodes.
Cytologic Patterns of Inflammation
Four main cytologic patterns are recognized in the cytologic examination of inflammatory exudates.
1. Inflammatory Cells Are Only Neutrophils
If neutrophils are degenerated there is a good chance that it is a bacterial infection. The sample should be observed for intranuclear bacteria, which usually are easy to find. This is the typical pattern of a superficial pyoderma.
In the case of sterile pustules neutrophils may be well conserved. Intact, hypersegmented neutrophils and acantholytic cells are very suggestive of pemphigus complex disease. Acantholytic cells are small, dark, round keratinocytes of the basal or spinous layer, which detach from the surrounding cells due to the rupture of intercellular bridges. A few acantholytic cells are sometimes observed in pustules due to bacterial infections. In this case they are less numerous, do not group easily in rafts, and bacteria are clearly visible inside the neutrophils. Bacteria together with acantholytic cells are also observed in samples from pemphigus diseases collected from under a crust, which has most probably been secondarily infected. In these doubtful cases, it is advised to obtain multiple skin biopsies and send them to a dermatopathologist for the definitive diagnosis.
Occasionally neutrophils (with or without secondary bacterial infection) may be observed together with other infectious agents, such as Demodex mites (in pustular demodicosis) and with fungal hyphae (in kerions).
2. Among the Inflammatory Cells There Are Macrophage
Macrophages are attracted to the inflammatory hearth some hours after the neutrophils. These cells are seen in deep pyoderma (very frequent in furunculosis), together with degenerated neutrophils and few intracellular bacteria. In this case neutrophils may still be the majority of the inflammatory cells and the inflammation is called pyogranulomatous.
If macrophages are > 50% of the cells, the inflammation is called granulomatous, and is usually associated with deep mycotic infections, atypical bacterial infections, leishmaniosis, foreign body reactions or sterile granulomatous diseases. In foreign body reactions multinucleated giant cells may be observed. Among the sterile granulomatous or pyogranulomatous diseases there are feline xanthomatosis, canine cutaneous and systemic histiocytosis, canine pyogranulomatous syndrome and the sterile nodular panniculitis. In every case of granulomatous or pyogranulomatous reactions pattern, if etiologic agents are not readily observed, a bacterial and a fungal culture from deep tissue samples should always be requested (also for atypical bacteria, such as acid-fast bacteria).
3. Among the Inflammatory Cells There Are Lymphocytes and/or Plasma Cells
Lymphocytes and plasma cells, together with other inflammatory cells (neutrophils and macrophages) are often seen in chronic lesions (longer than 7 days). If lymphocytes and plasma cells are dominating the cell population, there is a very strong antigenic drive, or a dysregulation of the immune system. Similar patterns are observed in immune-mediated diseases (feline plasmacellular pododermatitis) or in lymphomas. In any case a skin biopsy is necessary to determine the cause.
4. Among the Inflammatory Cells There Are Many Eosinophils
Eosinophils are rarely seen in dogs, and, if present, they are quite specific for one of the diseases mentioned above. On the contrary eosinophil are a common inflammatory cell in the cat, so their presence is significant only if in very high numbers.
In the dog eosinophils may be seen in bacterial furunculosis (as reaction pattern to the keratin as a foreign body), in eosinophilic nasal furunculosis (an arthropod bite triggered allergic reaction), in some cases of pemphigus foliaceus, in flea bite and in scabies papules, in the rare cases of eosinophilic granulomas (Husky predisposed) and in pustules in food allergic animals and in the rare condition called sterile eosinophilic pustolosis. In cats eosinophils are observed in eosinophilic plaques, collagenolytic (eosinophilic) granuloma, in some cases of indolent ulcus.
Cytology of the External Ear Canal
Cytologic preparations of a normal external ear canal may contain corneocytes, amorphous material (cerumen) and small amounts of Malassezia yeasts (< 5 / HPF). In case of Malassezia infection the number of yeasts is higher than 5–10 / HPF. In case of bacterial overgrowth a large number of bacteria is observed without inflammatory cells. In purulent otitis inflammatory cells (neutrophils) in large number are observed. Several bacteria are observed inside the neutrophils.
References are available upon request.