Clinically Pertinent Cytological Diff-Quick and Evaluation for the Reptilian Practitioner Kendal E Harr, DVM, MS, Dipl ACVP (Clinical ), 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 , 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 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 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 .

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 ; 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 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 . 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 with ) and pyogranulomatous inflammation.

Categories of inflammation

Suppurative-purulent-heterophilic inflammation characterized by >85% of WBCs present being heterophils. An acute inflammation process includes innate immunity where heterophils are the first inflammatory cell to migrate from the blood to damaged tissue. Typically a reaction to bacterial pathogens, but can also be associated with neoplasia, autoimmune processes, or response to chemical irritation.

Pyogranulomatous, or mixed inflammation, is characterized by >15% macrophages with the presence of hetero- phils and occasionally lymphocytes. Differentials include subacute bacterial infection, mycobacteria or nocardia, fungal infection, foreign body or injection reaction and furunculosis.

Granulomatous inflammation is characterized by >85% macrophages. Rarely seen cytologically, except in se- romas and fungal infections with low macrophage numbers.

Eosinophilic/ basophilic inflammation seen with parasitic migration and allergic or hypersensitivity reactions.

Bacteria, fungi and chlamydia

Abscesses due to a host of pathogenic and potentially commensal bacteria may occur in reptiles, especially those subjected to suboptimal temperatures as their immune system may literally be overgrown. Cytology should be used as a quality assessment of culture and sensitivity of samples. Cytology using Diff-Quik and Gram stain may also be used to guide initial therapeutic choices.

Mycobacterial rods: Refractile, unstained in Diff-Quik, Wright’s and Giemsa stains, but stain red with Kinyouns and other acid-fast techniques. Ziehl-Neelson techniques are used on histopathologic specimens but typically produce low yield samples in cytologic preparations.

Chrysosporium anamorph of Nannizziopsis (CANV): An aggressive and fatal necrotizing mycotic dermatitis. In bearded dragons

Candida: Can be visualized as single or budding yeast may also form pseudohyphae. Stains blue with Dif-Quik and dark blue with Gram stain.

Aspergillus: Septate fungal hyphae, have thin parallel walls, septa, and right angle branching. If well-aerated macroconidia and spores may be observed as rounded structures covered with oval to round fungal spores.

Cryptococcus neoformans: Round thick-walled yeast 5-20 µm in diameter with narrow-based budding. Its mu- copolysaccharide capsule forms a halo effect when stained.

Obviously, there are numerous other pathogens that should be investigated with culture and sensitivity and pos- sibly PCR.

Chlamydia: Obligate intracellular parasites that stain purple to blue with Diff-Quik and Giemsa. Gimenez, the confirmatory stain, stains elementary bodies red and cells green.

176 Building Exotics Excellence: One City, One Conference Viruses

Any intracytoplasmic inclusion that is suspected to be a viral inclusion should be investigated with electron microscopy and possibly polymerase chain reaction (PCR). Inclusion body disease in boas, pythons, and other snakes has been determined to be associated with an arena virus. This can be diagnosed based on identification on (CBC). An Inclusion Body Disease PCR is available.

Neoplastic Lesions

When evaluating a cytologic sample, differentiation of inflammation from neoplasia is key. Assessment of the WBCs present is crucial. Simple tumors lack inflammation and have monomorphic cell populations. Determine tissue of origin then assess malignancy.

Composed of distinct homogeneous cell populations, which don’t have inflammation (simple) or do have an inflammatory component (complex)

Differentiate benign or malignant neoplasms

Benign neoplasms have uniform cell populations with mature nuclei, little mitotic activity.

Malignant neoplasms show >3 criteria for malignancy:

• Anisocytosis

• Anisokaryosis

• Pleomorphism

• High or variable N:C ratio

• Increased mitotic activity

• Nucleoliosis

• Clumped chromatin

• Nuclear molding

• Multinucleation

Cells may contain unusual granules, pigment, or express unusual phagocytic activity. Occasionally cytologic morphology is not consistent with true malignancy.

Neoplasia cell types

Determine cell lineage based on characteristic morphologic features.

Epithelial neoplasms/carcinomas exfoliate in clumps or sheets (sometimes). Cells form tight cell junctions which then reveal clear lines and distinct cell borders

ExoticsCon 2015 Pre-conference Proceedings 177 Mesenchymal neoplasms/sarcomas are spindle cell tumours. Including fibromas, fibrosarcomas, hemangiomas, and hemangiosarcomas. These are typically of low cellularity as they exfoliate poorly, with individual cells or groups of cells in pink matrix that lack cell borders.

Round cell tumors most commonly found in reptiles include, lymphocytic leukemia/lymphoma, plasmacytomas, mast cell tumors, and melanomas. Histiocytic sarcoma has also been reported. These exfoliate well but may be difficult to diagnose on histopathology without special stain.

Melanomas are characterized by melanocytes. Melanomas have mass and hyperpigmentation does not. Melanin pigment granules are brown and must be differentiated from purple rod shaped bacteria.

Benign neoplasms

These neoplasms have uniform cytoplasm, nuclear size and shape and nucleoli and a low N:C ratio.

Xanthomatosis, benign tumors nodular or diffuse skin thickening indicating degenerating lipomas or after trauma. These are oily gritty aspirates contain free lipid, cholesterol clefts, vacuolated macrophages, and multinucleated giant cells. There are rare reports of xanthomas in the literature in reptiles and amphibians and these are not as common in reptiles as in birds. Similar diagnosis include degenerating or necrotic lipomas.

Lipomas, benign, greasy neoplasms of well-differentiated lipocytes. Lipid dissolves in methanol.

Cystic lesions

Cystic lesions with low cellularity, large amounts of debris or fluids, include parasitic cysts, severe trauma (se- roma), burns and ovarian cysts. Sebaceous cysts have been reported in lizards (iguanas).

Hemorrhagic lesions

Characterized by erythrocytes with variable numbers of macrophages. Erythrophagia indicates acute hemor- rhage differentiating it from blood contamination. Thrombocytes are not seen in hemorrhage after one hour. Siderophages may be seen and indicate more chronic haemorrhage. Hemorrhagic lesions include hematoma and neoplasia, esp. hemangioma and hemangiosarcoma.

Fluid Analysis

Complete fluid analysis should include total protein/total solids, total cell count or estimate (and possibly a dif- ferential cell count), and microscopic evaluation of morphology, infectious agents, and structures such as crystals.

Coelomic cavity

Normally there is minimal fluid in the coelomic cavity used for lubrication of organs and typically none can be aspirated in the normal reptile. Avoid enterocentesis, splenic and hepatic aspirate, and aspirates of coelomic fat pads.

Common cell types include low numbers of WBC and background blood contamination and mesothelial cells that line the body cavity. Mesothelial cells may be individual round cells or appear to be cohesive sheets of epithelium. These cells are phagocytic and may be reactive. Reactive mesothelial cells are notoriously misiden-

178 Building Exotics Excellence: One City, One Conference tified as neoplastic cells by the novice. When reactive, there is marked cytoplasmic basophilia with a light pink to purple hair-like corona surrounding the cell. Nuclei are round may have prominent nucleoli, bi and multi- nucleation, and exhibit frequent mitotic activity thus mimicking neoplastic cells. Lag chromatin in any cell type is pathognomonic for neoplasia.

Coelomic effusions should be categorized as transudates, modified transudates, and exudates as in mammals which give functional information regarding the disease process and leads to a different set of differential diagnoses. While these are the classic presentations, it should be noted that during disease states, reptiles can withstand remarkable dehydration that would kill many mammals and therefore protein values may be falsely increased. Alternately, in severe hepatic disease (for example with a component of hepatitis) albumin and other proteins cannot be produced and the protein concentration will be decreased in comparison to that expected in the disease process. There are iatrogenic influences that should be considered, such as fluid administration and other treatments.

Transudates are most frequently due to hypoproteinemia and loss of normal plasma oncotic pressure in the vasculature. Decreased production of albumin in the liver or increased loss through the kidney, gastrointesti- nal tract, or skin is the most common causes of transudate. Animals with dependent edema will typically have transudative effusion.

Modified transudates are the result of leakage from an inflamed vascular tree or vessel of normal integrity due to increased hydrostatic pressure. Therefore, these fluids have a higher protein content but still have lower cell content that is predominantly mononuclear (not heterophilic/ neutrophilic). Differential diagnosis includes cardiac disease, pulmonary disease, neoplasia, accumulation of lymph, and vasculitis (numerous etiologies).

Exudates are the result of an inflammatory process and are classified as septic (intracellular bacteria) or nonseptic. These fluids have high protein content and high cellular content.

Abnormal cytologic findings include cellular infiltrates with bacteria, fungi, egg yolk, or neoplastic cells. Egg yolk Effusion is yellow cream to clear or flocculent. Yolk is homogeneous to granular - globular, pink to blue, amorphous material containing macrophages, mesothelial cells, and multinucleate giant cells.

Neoplastic cells may be found in coelomic effusion and when found the interpretation, Neoplastic Effusion, should be rendered. Lymphocytic neoplasia may be diagnosed in effusions. Carcinoma cells may be found in coelomic cavity effusions due to ovarian/ granulosa cell, oviductal, or intestinal carcinoma. Differentiation of carcinoma cells from mesothelial cells is challenging even for the clinical pathologist. Direct aspiration of the mass may be required for definitive diagnosis.

Joint aspirates

Aspiration of the joint requires sterile preparation of the skin. Normal synovial fluid is viscous and poorly cellular, containing a dense pink background composed of mucopolysaccharides, few clasmatocytes (mononuclear cells) and rare erythrocytes due to contamination. Cells may be seen “windrowing” due to the highly proteinaceous nature of the fluid. Synovial fluid samples are classified as inflammatory or noninflammatory samples. Inflammation in synovial fluid indicates arthritis and infectious agents such as Mycoplasma sp. Noninflammatory lesions are most commonly the results of mineral (calcium and urate) deposits in the joint, differential diagnosis – degenerative joint disease found in aging, dietary imbalance (e.g. hypervitaminosis D), or chronic renal disease. Gout seen as chalky white material, or refractile uric acid crystals seen as needle-like configurations with polarized light.

ExoticsCon 2015 Pre-conference Proceedings 179 Gastrointestinal samples

Gastrointestinal (GI) samples include oral swabs, hepatic aspirates, gastric lavage, and cloacal swabs. Normal cell populations include squamous cells in the mouth and esophagus, and may, in some species extend into the stomach. Intestinal GI tract includes cuboidal to columnar epithelium but changes to squamous epithelium again near the anus. Tumors of the GI tract include papillomas and squamous cell.

Microsporidia are intracytoplasmic and common in bearded dragons.

Cryptosporidia are very common in leopard geckos found in intestinal cytology.

References

1. Alleman AR, Kupprion EK. Cytologic diagnosis of diseases in reptiles. Vet Clin N Am Exot Anim Pract. 2007 Jan;10(1):155-186.

2. Campbell TW, Ellis C. Avian and Exotic Animal Haematology and Cytology. 4th ed. Ames Iowa: Blackwell Publishing, 2015.

3. Casimire-Etzioni AL, Wellehan JF, Embury JE, Terrell SP, Raskin RE. Synovial fluid from an African spur- thighed tortoise (Geochelone sulcata). Vet Clin Pathol. 2004;33(1):43-46.

4. Harr KE, Henson KL, Raskin RE, Heard DJ, Phillips LA, Greiner EC. Gastric lavage from a Madagascar tree boa (Sanzinia madagascarensis). Vet Clin Pathol. 2000;29(3):93-96.

5. Heatley JJ. Evaluating reptile and amphibian hematology: A clinical perspective. Proc Annu Conf Assoc Avian Vet. 2014:39-46.

6. Jacobson ER, Reese DJ, Berry CR, Brock P, Agnew DW, Toplon DE, Abbott JR, Kridel HA, Alleman AR, Dunbar MD. What is your diagnosis? Granulosa cell tumor. J Am Vet Med Assoc. 2013;243(11):1533-1535.

7. Lafortune M, Göbel T, Jacobson E, Heard D, Brown D, Alleman R, Vliet K, Harr KE, Hernandez J. Re- spiratory bronchoscopy of subadult American alligators (Alligator mississippiensis) and tracheal wash evaluation. J Zoo Wildl Med. 2005;36(1):12-20.

8. Stenglein MD, Sanders C, Kistler AL, Ruby JG, Franco JY, Reavill DR, Dunker F, Derisi JL. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: Candidate etiological agents for snake inclusion body disease. mBio. 2012; 14:3(4):e00180-12. doi:10.1128/mBio.00180-12.

180 Building Exotics Excellence: One City, One Conference