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Clinically Pertinent Cytological Diff-Quick and Gram Stain Clinically Pertinent Cytological Diff-Quick and Gram Stain Evaluation for the Reptilian Practitioner Kendal E Harr, DVM, MS, Dipl ACVP (Clinical Pathology), April Romagnano, PhD, DVM, DABVP (Avian) Session #214 Affiliation: URIKA, LLC, Mukilteo, WA 98275, USA (Harr), Avian and Exotic Clinic of Palm City, Palm City, Florida and the Animal Health Clinic, Jupiter, FL 33458, USA (Romagnano). Abstract: The goals of this work were to: 1) improve knowledge of preanalytic sampling techniques including blood smears, fine needle aspirates, imprints, smears, and fluid preparation including oral, dermal and cloacal swabs, cystic and solid mass sampling, joint fluids and effusions, and fecal smears and floats; and 2) enable the reptilian practitioner to better identify basic cells, classify disease processes, as well as infectious agents such as bacteria, fungi, and other structures. Discussion of diagnoses and treatment will follow. Generalized disease processes cross species and classes. The most important rule for cytologic interpretation is to not overinterpret the cytologic findings. Cytology helps guide therapeutic decision making by classification of disease process as neoplasia, fungal infection, etc but may not provide a definitive diagnosis. One should only interpret to the correct level of diagnosis and know when to refer the cytology and biopsy the lesion. Preanalytical Blood collection Collect less than < 1% of a reptile’s body weight. Use heparinized, size appropriate pediatric microtainers or Capijects®. Use the jugular vein in species where possible as the large bore vein decreases the likelihood of lymph dilution common in samples from the caudal vein. The right jugular may be larger in some species of lizard and tortoise but the size difference is not as dramatic as in avian species. Both jugular veins may be used. Although the right jugular works well in chelonians and snakes, many reptiles can be safely bled from the tail. Other sites include the cervical sinus, basilic and medial metatarsal vein. Cytologic preparation Reptilian cells are larger than mammalian and avian cells, so larger gauge needles should be used if possible, depending on patient size to prevent shear forces and cell lysis. Humanitarian treatment of all species is paramount and undue harm and pain should not be caused. However, the practitioner should realize that most tumors and many inflammatory lesions may have decreased sensation due to necrosis or lack of innervation. ExoticsCon 2015 Pre-conference Proceedings 173 Consider anesthesia for restraint, analgesia, and staff and patient safety. The skin should be cleaned with an appropriate disinfectant for dermal commensals (chlorhexidine, other). Alcohol is ineffective for most dermal bacteria. Topical anesthetic may be injected around but NOT into the lesion as it can mimic edema and disrupt cellular morphology and staining. Preanalytical slide preparation is important. Clean preferably new glass slides should be readily available to make the smears as samples containing blood will clot quickly. All slides should be labelled with pencil or an ink that will not dissolve in methanol. Fine needle aspirates: Fine needle aspirates (FNA) may be used to sample solid tumors, cystic lesions or fluid. In reptiles, FNA is preferred in lesions with a fluid component. Some lesions may contain a fluid and solid com- ponent and a mix of FNA and core sampling may be required While fluid flows more freely, reptiles typically have caseated inflammatory debris which requires core sampling versus aspiration into a syringe. Firm, fibroelastic tumors such as those of mesenchymal origin (sarcoma) are also common in reptile species and also require large gauge (18) needles for sampling. Core sampling: Core sampling of solid masses will typically produce better intact samples than aspirates in reptiles. These may be accomplished with Jam Shidi biopsy needles, spinal needles, or the more typical 18-20 gauge venipunture needles. (Size should be adjusted to patient size). Stylettes should be used when pen- etrating skin to prevent skin contamination of the sample if the lesion is subdermal. Reptilian skin may clog the needle hub and prevent sampling. The needle is redirected through the lesion 4-6 times at different angles so that the cannula is filled with cells/tissue from the lesion to be sampled. Regardless of whether it is an aspirate, fluid or a core, the sample should be expelled from the needle hub using air drawn back in the syringe Skin scrapes: These scrapes are commonly performed to evaluate cutaneous lesions or dysecdysis in reptile species. This sampling can give valuable information as to type of inflammation and possible infectious agent but will not give information regarding deeper, underlying lesions. Also, it should be noted that some growth is expected in dead, sloughed epidermis/skin (detritus) and so low numbers of a heterogeneous population of bacteria and fungi may be interpreted as normal. The dull side of a scalpel blade is used to collect tissue. Tissue should be abraded until a small amount of blood is noted indicating complete epidermal sampling. Gently (with minimal pressure) scrape the blade edge across a clean glass slide to create a thin smear. Remember, if you can’t see through it, the pathologist will not be able to see through it when it is stained. For dry preparations of skin, small drops of mineral oil may be used adhere the tissue to the slide. Note this will dissolve in stain. Impression smears: These are collected by incisional or excisional biopsy may give a faster turnaround time to benefit patient and owners. The cut surface of the lesion (not the capsule) is blotted once on a clean paper and then gently imprinted several times across the slide to create at least one imprint of appropriate thickness. Swab preparations: These preparations may be made from oral lesions, fecal material, cloacal contents, tracheal material, draining tracts, conjunctiva, or nasal discharge. After the sample is collected, the swab is rolled along a glass slide 174 Building Exotics Excellence: One City, One Conference Hematology and cytology slides are thinly and uniformly smeared in the center of the slide as soon as possible after sample acquisition. Multiple slides should be made and submitted. Most labs will evaluate up to 4 slides for the initial price. Sample at the edge of smears may be not be stained and may never be evaluated at the laboratory. Standard push method blood smears, cover slip preparations, and pull preparations are acceptable in reptilian blood which tends to have less lysis than avian or fish samples. Hematology slides are stained with a Romanowsky stain; cytology and fecals can be stained with Romanowsky, Gram stain (bacteria), or Trichrome (parasitic evaluation). Hematologic description Red blood cells: Red blood cells (RBCs) are oval, large and nucleated in reptiles. Reptiles RBC counts vary inversely with reptile size and RBC size is largest in turtles, intermediate in snakes, and smallest in lizards. Seasonal and sex differences may be present in reptiles. Male reptiles have higher RBC counts then females. Heterophil: These are equivalent of the mammalian neutrophil. Round, oval or bilobed nucleus with pinkish granules, but these can be rod, oval, or elliptical shaped or may appear fused, melted or degranulated. Heterophils increase in stress hemograms and neoplasia and higher in aquatic chelonians. May decrease with leukopenia from infection, viral diseases such as inclusion body disease, low temperatures, and with hibernation. Lymphocyte: The nucleus is round with dense chromatin and high nucleus:chromatin (N:C) ratio, generally smaller than heterophils unless reactive with a basophilic cytoplasm. Increase with inflammation, wound healing and parasitic infection. Except for radiated or desert tortoises, most reptiles are lymphocytic, with lymphocyte numbers higher in summer and in females. Monocytes and Azurophils (in reptiles): The nucleus is round to kidney bean shaped with light staining cytoplasm often with vacuoles. Monocytes increase with chronic disease. Azurophils, most common in snakes, are large mono- nuclear cells with a fine dusting of pink to magenta cytoplasmic granules. Monocytes and azurophils are similar. Eosinophil: The nucleus is lobed in a round cell with pink round plump distinct granules; can increase with allergy and parasitism. Some reptile species have granules that stain blue. Eosinophilia is lowest during hiber- nation. Some reptiles are eosinophilic, ex: green sea turtles. prehensile tailed skink. Lizards have low numbers and some snakes lack them. Basophil: The nucleus is round to oval with deeply blue staining round granules. Basophils are rare in sea turtles, but high in Reeve’s turtles, snappers and cooters. Cytology Evaluates cell morphology and differentiates the physiologic process of inflammation from cysts and neoplasia. With cytology a positive is positive. A negative means it wasn’t present in the sample. Histopathology unlike cytology evaluates architecture of the lesion, not cellular morphology. Inflammatory and infectious lesions Reptile inflammation characterized by increased numbers of white blood cells (WBCs) over the blood present in the background. In general infectious agents in reptiles include bacteria, fungi, parasites, Chlamydia spp. and ExoticsCon 2015 Pre-conference Proceedings 175 viruses. The two most common types of inflammation found cytologically in reptiles are heterophilic inflamma- tion (not to be confused
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