C O N F E R E N C E 17 10 February 2016

Joint Pathology Center Veterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2015-2016

C o n f e r e n c e 17 10 February 2016

Casey J. LeBlanc, DVM, PhD Diplomate, ACVP Clinical Pathologist & CEO KDL VetPath Knoxville, TN & Springfield, VA

CASE I: TAMU-1-2013 (JPC 4033379).

Signalment: 18-day-old thoroughbred colt (Equus caballus)

History: A healthy newborn foal nursed and the next day, became lethargic and had hemoglobinuria. With progressive stupor, icterus, hyperbilirubinemia and anemia, the foal was presented to the clinic at 2 days of Mucous membranes, foal. The mucous membranes of this foal are extremely yellow, indicating icterus. (Photo age. courtesy of: Dept Vet Pathobiology, College Vet Med Texas A&M University, College Station, TX 77843-4467 Gross Pathology: Yellow mucous http://vetmed.tamu.edu/vtpb) membranes and tissues (icterus); ~200ml abdominal transudate with fibrin strands; Total bilirubin 14.5 mg/dl (0-1.9) myocardial mottling grey and red GGT 124U/L (0-53) (hemorrhage and necrosis) a small Thebsian Alkaline phosphatase 553U/L (128-512) vein in the noncoronary aortic sinus; urachus ALT 850U/L (134-643) patent. Saline agglutination test positive Mare serum Anti-Qab positive Laboratory Results: PCV=12 (32-53) no Nrbc’s Histopathologic Description: The section Platelets 470,000/ul (100-350,000) of liver has widespread atrophy of TP 4.7g/dl (5.3-7.3) hepatocytes with hypertrophied and

Liver, foal. There is extensive pigment deposition throughout the section, to include brown granules in hepatocytes (lipofuscin- yellow arrows), brown spicular material (acid hematin – green arrows) and distended bile canaliculi (cholestasis – black arrows). Syncytial hepatocytes, often with peripheralized nuclei are scattered throughout the section. (HE, 40X) occasionally binucleate nuclei. Some Contributor’s Morphologic Diagnosis: hepatocyte syncytia contain 6-8 nuclei. Often, the hepatocytes exhibit feathery Liver: Subacute hepatopathy with centrilo- degeneration, but centrilobular hepatocytes bular hepatocellular degeneration, necrosis are degenerating or have individual cell and collapse; edema, lipofuscinosis, bile necrosis. The spaces of Disse are expanded casts and mild bile duct proliferation; (edema), and sinus leukocytosis is common hepatocyte syncytia; erythrophagocytosis with leucocytes often concentrated in areas and hemosiderosis; multifocal neutrophilic of hepatocyte loss. Scattered lipofuscin- hepatitis. laden macrophages are often near the pale staining triads, and bile casts and mild bile Contributor’s Comment: This is a classic duct proliferation are noted. Throughout the case of equine neonatal isoerythrolysis section are syncytia similar to Langhan’s /isoimmune hemolytic anemia (NI). The type multinucleate giant cells with nuclei animal was treated with dexamethasone, surrounding a tan, granular cytoplasm banamine, and antibiotics; clinical and post- (lipofuscin). Erythrophagocytosis by mortem blood cultures were sterile. No Kupffer cells is noted, and iron-staining organisms were noted with Gram or GMS Kupffer cells are in sinusoids. Hepatic cords stains. While there is inflammation is in the are disassociated (shock). lesion, it was felt to reflect secondary, cholestasis-induced hepatitis.

When reviewing the case with the resident, growth factors.7 Just as in autoimmune the pathologist commented that the giant hemolytic anemia in early childhood, cells were classically seen with NI; syncytial cell hepatopathy has been reported however, the resident noted that this lesion in cases of equine NI.2 It was noted in a case is not mentioned in textbooks as a lesion of NI presented in the WSC of 5-14-86. associated with NI. Young horse How does one explain the lack of the lesion hepatocytes often form syncytia, and giant description? Foal livers in fatal cases of NI cell hepatopathies/ hepatidides are are “busy,” and the syncytial cells, while described; especially associated with lepto- typical, may be down-played in face of the spirosis,13,20 but “idiopathic” hepatocyte catastrophic clinical and autopsy findings. syncytia have been described in 5-7 month- Because our cases are often referral cases old, equine abortuses3 and have been seen and are subacute to chronic, it may also be by this contributor in leptospira PCR- that syncytia are just more frequent in long- negative cases. In human infants with standing, fatal cases. Syncytia are jaundice, it was described as “giant cell characteristic. Foals seem prone to respond hepatitis”17 or “syncytial giant cell this way. Although neonatal infections may hepatitis.15 Initially, some considered it an complicate clinical NI, infectious agents are expression of viral hepatitis, particularly not always documentable. We should serum hepatitis, in infancy. Some feel the remember that only recently a viral agent of lesion reflects a specific insult such as blood equine Theiler’s disease has been group incompatibility between mother and identified,4 which may play a role (though I infant. It has been considered a nonspecific doubt it). response of liver regeneration to any injury.10,19 Still others consider it a Kernicterus is always a concern in cases of congenital defect in the formation of bile prolonged unconjugated hyperbilirubinemia canaliculi or another genetically transmitted in neonates. This foal had no macroscopic abnormality.5,14 Popper and Schaffner14 lesions of kernicterus, the cortex did not concluded hepatocyte giant cell formation fluoresce and no neuronal lesions were was a feature of cholestasis in infancy, and noted in the cerebral cortex, Purkinje cells or that it may be associated with some inflam- hippocampus, where it has been described in matory changes. Syncytial cell hepatitis has foals with NI.12 It’s of interest that syncytia been reported in humans and is associated were not reported in any of their autopsy with bacterial sepsis (especially cases, even their chronic cases. There is a toxoplasmosis, syphilis listeriosis, case report of NI in a three-day-old foal with tuberculosis), viral diseases (cyto- kernicterus and neuronal necrosis megalovirus, herpes simplex, varicella, represented macroscopically by yellow- echovirus, parvovirus B19, enterovirus, discolored nuclei.6 rubella, human herpesvirus-6, human immunodeficiency virus, hepatitis types A, For completeness, it is interesting that this B and C, Marburg virus and paramyxo- colt had a persistent neutrophilia and virus), liver transplantation and death from developed a thrombocytosis. Alloimmune, severe liver failure.8,16 However, in the neonatal neutropenia has been described in a neonatal period it is considered a non- foal with Ka antibodies in a recent report of specific reaction of immature hepatocytes to NI.21 The most complete study of anti- various forms of “aggression,”10 and the erythrocyte antibodies in mare serum and syncytia stain with nuclear proliferation and colostrum was done in thoroughbred and

from opsonization of red blood cells by complement component C3b, or by antibody, followed by phagocytosis.18 Lipofuscin is an intracellular pigment thought to accumulate as a result of aging and cellular “wear and tear,” including processing of senescent cellular organelles and other material. It accumulates within lysosomes most significantly in post mitotic or slowly dividing cells as an end product of autophagy and is often seen as a perinuclear golden brown pigment. It looks very similar histologically to ceroid, which is more Liver, foal: There are moderate amounts of iron within Kupffer cells. (Perl’s, 40X) commonly associated with pathologic conditions such as severe malnutrition and vitamin E deficiency or can be seen in standardbred mares and Qa and Aa seemed certain inherited conditions such as neuronal to be the most common alloantibodies found 1 ceroid lipofuscinosis (see WSC 2015-16, at large; however, these alloantibodies may conference 10, case 1). Ceroid is known to not be the most common seen in clinical 2, 9 accumulate in both Kupffer cells and cases of NI. Alloantibodies to Qa antigens hepatocytes as well as other tissues and can were found in the mare of our case. The colt be intracellular or extracellular. Lipofuscin of our study did not show a regenerative is thought to accumulate slowly over the life erythroid response. Over the 2-week of the animal, whereas ceroid usually hospitalization, no nucleated erythrocytes accumulates rapidly depending on the were noted and the bone marrow had associated condition. Histochemical tech- erythrocytic hypoplasia. We wondered if niques which can aid in identifying both this may have reflected a specific ceroid and lipofuscin include Sudan black, destruction of precursors of the erythroid oil-red-O, PAS and Ziehl-Neelsen stains. series. Although differences in content of the two pigments have been demonstrated by special JPC Diagnosis: Liver: Hepatocellular histochemical techniques, it is not possible degeneration and atrophy, diffuse, severe to differentiate the two pigments in standard with Kupffer siderosis, cholestasis, and H&E stained sections.11 hepatocellular syncytial cell formation. The primary histologic features in this case include hepatocyte degeneration and Conference Comment: Neonatal iso- atrophy, with formation of syncytial cells. erythrolysis is a type II hypersensitivity Extensive pigmentation of hepatocytes and reaction that results from antibodies directed Kupffer cells was present throughout the against neonatal red blood cells, most section prompting extensive discussion. . frequently IgG or IgM. This leads to The brown granular to globular intracellular activation of complement component C1q pigment within hepatocytes was interpreted and eventual formation of the membrane as lipofuscin and within Kupffer cells as attack complex which lyses the red blood hemosiderin. An iron stain confirmed the cell. Red blood cell lysis can also result presence of moderate amounts of hemo-

4 siderin within Kupffer cells and occasionally mule foal: 18 cases (1988-2003). J Am Vet within hepatocytes. Viewing under fluor- Med Assoc. 2005; 227: 1276-1283. escence confirmed the presence of small amounts of autoflourescent pigment within 3. Car BD, Anderson WI. Giant cell hepatocytes consistent with lipofuscin. hepatopathy in three aborted midterm equine Abundant birefringent, yellow-brown spi- fetuses. Vet Pathol. 1988; 25:389-91. culated material was ultimately identified as 4. Chandriani S, et al. (2013) Identification acid hematin. Numerous dilated bile canal- of a previously undescribed divergent virus iculi were also noted, indicating cholestatic from the Flaviviridae family in an outbreak disease. A Hall’s stain for bile confirmed of equine serum hepatitis. ProcNatl Acad this finding. Sci U S A. 2013 Apr 9; 110(15):E1407-15.

doi: 10.1073/pnas.1219217110. We thank the contributor for providing clinical pathology data with the submission, 5. Clayton PT, Disorders of bile acid which greatly adds to the teaching value of synthesis. J Inherit Metab Dis. 2011; the case. One of the best indicators of 34:593-604. regenerative anemia is reticulocyte count, but horses do not release reticulocytes. The 6. David JB, Byers TD, Braniecki A, absence of nucleated red blood cells may Chaffin MK, Storts RW. Kernicterus in a suggest a nonregenerative anemia; however, foal with neonatal isoerythrolysis. Comp the best way to determine regenerative status Cont Educ Prac Veterinarian. 1988; 20:517- would be a bone marrow sample. In this 20. case, a bone marrow aspirate revealed erythrocytic hypoplasia, as indicated above 7. Fang JWS, González-Peralta RP, Chong in the contributor’s comment. Conference SKF, Lau GM. Hepatic expression of cell participants briefly discussed bilirubin proliferation markers and growth factors in metabolism and the elevated total bilirubin giant cell hepatitis: Implications for the in this case was interpreted as both pre- pathogenetic mechanisms involved. JPGN. hepatic and hepatic, which fits with both the 2010; 52:65-72. pathogenesis and histologic findings. 8. Hicks J, Barrish J, Zhu SH. Neonatal syncytial giant cell hepatitis with Contributing Institution: paramyxoviral-like inclusions. Dept Vet Pathobiology, College Vet Med Ultrastructural Pathol. 2001; 25:65-71. Texas A&M University 9. MacLeay JM. Neonatal isoerythrolysis http://vetmed.tamu.edu/vtpb involving Qc and Db antigens in a foal. J Am Vet Med Assoc. 2001; 219:79-81. References: 10. Maggiore G, Sciveres M, Fabre M, Gori 1. Bailey E. Prevalence of anti-red blood L, et al. Giant cell hepatitis with auto- cell antibodies in the serum and colostrum immune hemolytic anemia in early of mares and its relationship to neonatal childhood: Long-term outcome in 16 isoerythrolysis. Am J Vet Res. 1982; children. J Pediat. 2012; 159:127-132. 43:1917-21. 11. Myers RK, McGavin MD, Zachary JF. 2. Boyle A, Magdesian KC, Ruby RE. Cellular adaptions, injury and death: Neonatal isoerythrolysis in horse foals and a Morphologic, biochemical and genetic

5 bases. In: McGavin MD, Zachary JF, eds. leptospiral infection. Can Vet J. 1988; Pathologic Basis of Veterinary Disease. 5th 29:1003-4. ed. St. Louis, MO: Mosby Elsevier; 2012:43-44. 21. Wong DM, Alcott CJ, Clark SK, Jones DE. Alloimmune neonatal neutropenia and 12. Polkes AC, Giguere S, Lester GD, Bain neonatal isoerythrolysis in a Thoroughbred FT. Factors associated with outcome in foals colt. J Vet Diag Invest. 2013; 24: 219-226. with neonatal isoerythrolysis (72 Cases, 1988 –2003). J Vet Intern Med. 2008; 22:1216-1222.

13. Poonacha KB, Smith BJ, Donahue JM, CASE II: A15-9631 (JPC 4065767). Tramontin RR. Leptospiral abortion in horses in central Kentucky. Proc 36th Ann Signalment: 1-year-old, gelding, Arabian Convention Am Asso Eq Practitioners. 1991; 36:397-402. horse (Equus ferus caballus) 14. Popper H, Schaffner F. Pathophysiology History: Forty days after being gelded, of cholestasis. Hum Pathol. 1970; 1:1-24. vaccinated (annual vaccines plus tetanus antitoxin), and de-wormed (ivermectin), this 15. Portenza L, Luppi M, Barozzi P, Rossi horse was acutely anorectic and ataxic in the G. HHV-6 in syncytial giant cell hepatitis. morning with progression to recumbency by NE J Med. 2008; 359:593-602. afternoon. On physical examination at the Veterinary Teaching Hospital later that day, 16. Raj S, Stephen T, Debski RF. Giant cell the horse was recumbent in the trailer, hepatitis with autoimmune hemolytic depressed but responsive, and hypothermic anemia: A case report and review of (92.2 F). Other abnormal physical pediatric literature. Clin Pediatr. 2011; examination findings included hyperpnea 50:357-9. (28 breaths per minute), intermittent vertical 17. Shaffner F, Popper H. Morphologic nystagmus, bilateral inconsistent menace studies in neonatal cholestasis with response, mydriasis and delayed pupillary emphasis on giant cells. Ann NY Acad Sci. light reflex, prolonged capillary and jugular 1963; 111:358-374. refill times, icterus, and dehydration.

18. Snyder PW. Diseases of immunity. In: Gross Pathology: Icterus, hemoabdomen, McGavin MD, Zachary JF, eds. Pathologic perirenal hemorrhage, subendocardial Basis of Veterinary Disease. 5th ed. St. hemorrhage Louis, MO: Mosby Elsevier; 2012:263-264. Liver—pale yellow-brown, flaccid, 2.3 Kg (0.99% body weight) 19. Torbenson M, Hart J, Westerhoff M, Azzam RK. Neonatal giant cell hepatitis: Histological and etiological findings. Am J Laboratory Results: Surg Pathol. 2010; 34:1498-1503. Test Result Reference Range 20. Wilke IW, Prescott JF, Hazlett MJ, Packed cell 58% 35-50% Maxie MG, van Dreumel AA. Giant cell volume (PCV) hepatitis in four aborted foals: A possible Segmented 0.4 x 6.0-12.0 x neutrophils 103/μL 103/μL

Total Protein 7.6 g/dL 5.7-8.1 PCR for Theiler’s disease-associated g/dL flavivirus (TDAV), Cornell University Lactate 7.2 <2 mmol/L Animal Health Diagnostic Center: mmol/L nNegative. Fibrinogen 112 115-289 mg/dL mg/dL Glucose <20 73-124 Histopathologic Description: Swelling, mg/dL mg/dL lysis, and drop-out of hepatocytes affected Blood urea 3 mg/dL 8-27 mg/dL all hepatic lobules and were centrilobular to nitrogen (BUN) massive. Hepatocytes in lobular centers Creatinine 2.0 0.6-1.8 were generally not recognizable. In the mg/dL mg/dL lobular periphery, where viable parenchyma remained, hepatic plates were disrupted and TCO2 19 23-31 mmol/L mmol/L hepatocytes had vacuolated cytoplasm. Brown crystals were in the cytoplasm of Anion gap 26.4 12-20 periportal hepatocytes. Brown granular mmol/L mmol/L pigment was in Kupffer cells. Inspissated Aspartate 3,563 206-810 bile was observed in a few canaliculi. There aminotransferase IU/L IU/L was patchy increase in fibrous tissue and (AST) mononuclear leukocytes (mainly lymph- Alkaline 933 IU/L 109-331 ocytes) in portal tracts, but neither phosphatase IU/L inflammation nor fibrosis was severe. Bile γ-Glutamyl 158 IU/L 12-46 IU/L duct proliferation was not appreciated. transferase (GGT) Creatine kinase 9811 88-453 Changes in the cerebrum (slide not (CK) IU/L IU/L submitted to WSC) included increased space Bilirubin 21.1 0.10-2.60 around vessels and cortical neurons with mg/dL mg/dL Alzheimer type II astrocytes (hypertrophied and hypochromatic nuclei). These changes Unconjugated 15.3 were considered consistent with hepatic mg/dL encephalopathy. In addition, many deep cortical neurons had ischemic change with a Conjugated 5.8 shrunken angular profile, intense cyto- mg/dL plasmic eosinophilia, and pyknosis. Blood ammonia 254.4 25.0-75.0 μmol/L μmol/L Contributor’s Morphologic Diagnosis: Aerobic and anaerobic bacterial culture: no Submassive hepatic necrosis significant growth.

against African horse sickness with an equine antiserum-containing live virus vaccine.6 Today, Theiler’s disease is usually recognized in horses 4-10 weeks after injection with a product that contains equine serum or plasma, though some affected horses have no history of injection with equine blood-containing biologic products. Although the incubation period is long (42- 90 days), clinical disease (subclinical cases are also recognized) is fulminating with acute hepatic failure and death within 24 hours in up to 90% of clinically ill horses. Liver, horse. The retiform pattern noted at subgross At autopsy, the carcass is icteric. The liver results from diffuse centrilobular and midzonal hepatocellular necrosis (pale areas), contrasted with the is generally close to normal size, but flaccid remaining viable periportal hepatocytes, which stain more (“dish-rag” liver) due to massive necrosis intensely. (HE, 4X) with loss of so many hepatocytes. The histologic lesion is centrilobular to massive Contributor’s Comment: The laboratory necrosis with mild, mainly lymphocytic, results—especially hypofibrinogenemia, inflammation of portal tracts. Surviving decreased BUN, hypoglycemia, elevated periportal hepatocytes are swollen with hepatic enzyme activity, hyperbilirubinemia, vacuolated cytoplasm. and hyperammonemia—and the history and clinical signs all pointed to liver disease Theiler’s disease has long been suspected to with hepatic encephalopathy as a likely be a viral hepatitis, but the first candidate explanation for the neurologic signs. The virus, Theiler’s disease-associated virus initial differential diagnosis included (TDAV, a pegivirus in the Flaviviridae Theiler’s disease (equine serum hepatitis), family), was only identified a few years ago pyrrolizidine alkaloid or other hepa- in horses treated with equine antiserum to totoxicosis, ascending cholangiohepatitis, botulinum toxin.1 Another pegivirus called chronic hepatitis, and hepatic steatosis. equine pegivirus has been identified in horses, but has not been documented to Postmortem gross and histologic lesions cause hepatitis.4 The recently discovered were typical of Theiler’s disease, hence the non-primate hepacivirus (NPHV, equine PCR testing for Theiler’s disease-associated hepacivirus) is another member of the virus (TDAV). This horse was negative by Flaviviridae family that is hepatotropic and PCR for TDAV, but the serum was positive can result in transient or chronic infection for a previously unreported non-flavivirus with elevated hepatic enzymes and hepatitis that is under investigation (personal comm- in horses.3-5 Many horses (30-40%) have unication, Drs. Bud Tennant, Tom Divers serum antibodies to NPHV; fewer horses and colleagues at Cornell University and have detectable viral RNA in the peripheral Columbia University). blood. The horse has been proposed as an animal model of viral hepatitis because Equine serum hepatitis or Theiler’s disease NPHV is so closely related to the human was first described in 1919, when Arnold hepatitis C virus. Theiler reported acute hepatic atrophy and hepatitis in South African horses vaccinated

Liver, horse. Higher magnification demonstrating the differential staining between hepatocytes in centrilobular and midzonal areas and those in the periportal areas. (HE 80X)

JPC Diagnosis: Liver: Necrosis, centrilo- disease. Lesions range from the presence of bular to midzonal, diffuse with hepa- abundant degenerate and/or necrotic tocellular lipidosis. hepatocytes to complete loss of parenchymal cells. Many hepatocytes are often Conference Comment: Although the completely lost, leaving behind variable majority of cases of equine serum hepatitis numbers of degenerate and lipid-filled are associated with prior administration of hepatocytes, dilated congested sinusoids, equine biologics, cases have also been and collapsed stromal remnants. Hem- documented in horses with no history of orrhage and acute necrosis are not typical prior injection. While Theiler’s disease- findings. Other common features, albeit associated virus has been proposed as an 1 varying in severity, include low numbers of etiology for this condition, additional inflammatory cells, mild fibrosis in portal research is necessary to prove definitive 2 2 areas and an increase in bile duct profiles. causation and fulfill Koch’s postulates. It is also unclear what percentage of horses In this case, conference participants develop subclinical hepatic disease after described diffuse loss of hepatic cord exposure to equine origin biologics as the architecture with hepatocellular deg- disease is rarely diagnosed prior to eneration, necrosis and loss. There is development of hepatic failure; however, stromal collapse and mild portal bridging some animals have been known to survive fibrosis. The brown pigment present within after mild disease. Histologic findings often Kupffer cells and hepatocytes was suggest a subacute process, which contrasts interpreted as hemosiderin. The section is with the relatively acute clinical course of

9 moderately autolytic and there is abundant References: acid hematin present. 1. Chandriani S, Skewes-Cox P, Zhong W, We thank the contributor for providing Ganem DE, et al. Identification of a clinical pathology data with the submission, previously undescribed divergent virus from which greatly enhances the teaching and the Flaviviridae family in an outbreak of learning value of the case. Severe equine serum hepatitis. 2013. Proc Natl neutropenia is most commonly associated Acad Sci USA. 2013; 110(15):E1407-E1415. with endotoxemia, but the preciseetiology is 2. Cullen JM, Stalker MJ. Liver and Biliary difficult to ascertain in this case. Elevated System. In: Maxie MG, ed. Jubb, Kennedy, packed cell volume (PCV) is likely and Palmer's Pathology of Domestic secondary to dehydration and the normal Animals. 6th ed. Vol 2. St. Louis, MO: total protein is indicative of hypo- Elsevier; 2015:312-313. proteinemia in a dehydrated animal. Low fibrinogen, blood urea nitrogen, elevated 3. Pfaender S, Cavalleri JMV, Walter S, ammonia and low glucose are indicators of Doerrbecker J, et al. Clinical course of liver failure. The profoundly low glucose is infection and viral tissue tropism of hepatitis not compatible with life and may indicate C virus-like nonprimate hepaciviruses in some degree of artifact. The decreased horses. Hepatology. 2015; 61(5):447-459. TCO2 and elevated anion gap are indicative of a metabolic acidosis and the unmeasured 4. Ramsay JD, Evanoff R, Wilkinson TE, anions in this case include lactate and renal Divers TJ, et al. Experimental transmission acids. Elevated AST is secondary to both of equine hepacivirus in horses as a model myocyte and hepatocyte damage and for hepatitis C virus. Hepatology. 2015; elevated CK provides additional evidence of 61(5):1533-1546. muscle damage. Elevated GGT and ALP are both indicators of cholestasis and the 5. Scheel TKH, Kapoor A, Nishiuchi E, most common cause of elevated total Brock KV, et al. Characterization of bilirubin horses is anorexia. The hyper- nonprimate hepacivirus and construction of bilirubinemia in this case is higher than can a functional molecular clone. Proc Natl be attributed to anorexia alone and, Acad Sci USA. 2015; 112:2192-2197. therefore; the remarkable hepatic changes 6. Stalker MJ, Hayes MA. Liver and likely contribute to the elevation. Elevated biliary system. In: Maxie MG, ed. Jubb, bilirubin can also be seen in cases of Kennedy, and Palmer’s Pathology of endotoxemia due to impaired excretion of Domestic Animals. 5th ed. Vol. 2. Elsevier, conjugated bilirubin into the biliary tract. St. Louis, MO; 2007:297-388.

Contributing Institution: Purdue University CASE III: 705-14 (JPC 4066679). Animal Disease Diagnostic Laboratory: http://www.addl.purdue.edu/ Signalment: 10-year-old, neutered female, Department of Comparative Pathobiology: dog, Pekingese (Canis familiaris) http://www.vet.purdue.edu/cpb/ History: The dog was presented to the veterinary hospital with a history of apathy and severe anemia. Due to a previous

10 diagnosis of hemolytic anemia, the dog was thickened by fibrosis and with several received prednisolone (10 mg BID) and whitish plaques (1.0 to 3.0 mm in thickness) doxycycline (5 mg/kg IV bid), for two of chronic active inflammation (perisplenitis weeks, and a blood transfusion was with multifocal hyperemia). On the cut performed 40 days prior to admission. One surface, the splenic parenchyma protruded week after the admission, a blood sample and it was firm (carnous) and reddish, with was collected for total blood cell count and multiple small white foci. The liver was

serological tests for detecting anti- diffusely pale red with multifocal white foci (1.0 mm in diameter) in the parenchyma. The lungs were moderately congested and edematous. Mild myxomatous valvular degeneration was observed in the mitral valve. There were several petechiae in the epicardium of left ventricle and in the peritoneum. Dark red areas (approximately 2.0 cm in diameter), with sharp edge were observed in the parenchyma of the right and left kidney (acute infarction).

Laboratory Results: The initial serological tests resulted in positive indirect immuno- Spleen, dog. The spleen parenchyma is expanded by large numbers of macrophages, effacing the normal red fluorescence reaction (RIFI) (1:40 dilution), and white pulp architecture. (HE, 5X). and negative by ELISA, whereas the second serological tests were positive by both Leishmania antibodies. At physical techniques (i.e. RIFI and ELISA). examination, the cornea of the left eye was diffusely opaque, and there was moderate The dog was observed for two months and exophthalmos. Ophthalmic examination total blood cell count analyses were revealed retinal detachment. The clinical performed weekly. Overall, the dog had condition of the dog was followed-up for marked anemia, leukopenia, and thromb- two months, and total blood cell count was ocytopenia. Clinical pathology results are performed weekly. During this period, the presented in Table 1. dog received three blood transfusions. The After the last transfusion, i.e. 62 days after animal died two and half months after the the first hematological evaluation, the RBC, admission, despite medical treatment. HCT, and hemoglobin values were within the reference range. Gross Pathology: The dog was in good Concomitantly with the fourth hem- body condition. The cornea of the left eye atological evaluation, a bone marrow was diffusely opaque and whitish. There aspiration was performed. It was noticed the was moderate exophthalmos of the left eye. paucity of hematopoietic precursors, mainly The upper and lower incisor teeth were from the erythroid lineage. Serological tests absent. The cervical, popliteal and at this time resulted in positive ELISA and mesenteric lymph nodes were enlarged. On negative RIFI. the cut surface, these lymph nodes were All hematological analyses revealed reduced diffusely light brown and soft (interpreted as numbers of platelets and lymphocytes. The hyperplasia and hemosiderosis). The spleen numbers of monocytes remained below the was markedly enlarged. The splenic capsule

Occasional areas of neovascularization are within the capsule. There are scattered areas of karyorrhectic and cellular debris (lytic necrosis) containing occasional viable and degenerate neutrophils admixed with variable amounts of an eosinophilic beaded to fibrillar material (fibrin). There is multifocal histiocytic erythrophagocytosis, rare megakaryocytes, and numerous macrophages containing intra- Spleen, red pulp. The red pulp is replaced by large numbers of amastigote-laden cytoplasmic granular macrophages admixed with numerous plasma cells and fewer lymphocytes. Moderate and brown pigment numbers of macrophages (arrows) contain phagocytized erythrocytes. (hemosiderosis). reference range in most of the hematological Bone marrow (proximal femur): The exams. marrow parenchyma is completely replaced (myelopthisis) by numerous macrophages, Histopathologic Description: Spleen: The moderate plasma cells and fewer normal splenic architecture is markedly lymphocytes. Macrophages contain myriad distorted due to complete replacement of the of basophilic structures similar to those red and white pulp by numerous described in the spleen (Fig. 4). macrophages, moderate numbers of plasma Multifocally, are areas of karyorrhectic and cells, fewer lymphocytes, and occasional cellular debris (lytic necrosis) containing neutrophils, that expand the cords, fill the occasional viable and degenerate neutrophils sinuses and invade the splenic trabeculae. admixed with variable amounts of an Macrophages are enlarged and contain eosinophilic beaded to fibrillar material myriad of 2.0-4.0 m, ovoid, intra- (fibrin). There is multifocal hemosiderosis. cytoplasmic amastigotes (Fig. 3) with a 1.0 Tissues not submitted: m nucleus, surrounded by a 1.0-2.0 m clear zone and a kinetoplast perpendicular to Lymph nodes present changes similar to the nucleus (compatible with amastigotes of those described in the spleen. Additionally, Leishmania spp.). Multifocal areas of the moderate lymphoid hyperplasia and capsule are expanded due to deposition of increased plasma cell differentiation are variable amounts of fibrous connective observed. Macrophages containing myriad tissue and infiltration of scattered lymph- of amastigotes of Leishmania spp. are seen ocytes, plasma cells, and macrophages. within lymphatic sinus, medullary cords,

12 cortical and paracortical regions. In the liver Africa. In these former regions, transmission there are multiple areas with loss of is mostly anthroponotic.9 In Latin America, hepatocytes and infiltration of macrophages, approximately 90% of the cases of human plasma cells and lymphocytes. Some visceral leishmaniosis are reported in Brazil, macrophages are loaded with amastigotes of and occur in almost all of the states of the Leishmania spp. In both organs there is country. The higher incidence of the disease moderate multifocal hemosiderosis. in humans takes place in the northeast region of the country (65% of the cases), The kidneys presented with moderate followed by the southeast (14%) north membranous glomerulonephropathy. The (14%) and centerwest (14%).3,6 Currently, Lumina of several tubules are ectatic, and the disease is gradually spreading to the contain aggregates of eosinophilic material south and west regions of the country.3,10 (protein casts). Studies comparing the incidence or prevalence of the disease in dogs in different Amastigotes of Leishmania spp. were regions of the country are scarce. In Belo detected by immunohistochemistry Horizonte, capital of the state of Minas (Streptavidin-biotin-peroxidase) in the Gerais, CanL is considered endemic with spleen (Fig. 5) and in the bone marrow (Fig. increasing seroprevalence in the canine 6 and 7). population. From 1994 to 2000, this prevalence was estimated in 3.6% of all dogs of the county. In 2007, this index Contributor’s Morphologic Diagnosis: increased to 9.3%.3,8

Spleen: Splenitis and perisplenitis, The life cycle of Leishmania spp. involves histiocytic and plasmacytic, diffuse, severe, two hosts – a phlebotomine sand fly vector with myriad of intrahistiocytic amastigotes, (genus Lutzomyia in the New World) and a etiology consistent with Leishmania spp. mammal (including rodents, canids, or humans). Leishmania spp. occurs as Bone marrow: Myelitis, histiocytic and flagellated, extracellular promastigotes in lymphoplasmacytic, diffuse, severe, with the gut of sand fly vectors. Infection occurs myriad of intrahistiocytic amastigotes, when a feeding sand fly deposits metacyclic etiology consistent with Leishmania spp. promastigotes into the dermis of the host. In mammalian hosts, Leishmania spp. occur as Contributor’s Comment: Canine amastigotes (2.0 to 3.0 m in diameter) leishmaniasis (CanL) is a major global within mononuclear phagocytes in the skin, zoonosis, potentially fatal to humans and 11 bone marrow, and visceral organs. dogs. Dogs are the main reservoir of the Although vector-borne is the most important infection to humans.3 The disease is route of transmission, CanL can also be endemic in more than 70 countries in the transmitted in the absence of the invertebrate world and occurs predominantly in the 13 vector. southern Europe, Africa, South and Central

America.11 While Leishmania infantum CanL is manifested by a broad spectrum of (synonym L. chagasi) occurs in Latin clinical signs and degrees of severity. In the America, as well as in the Mediterranean typical CanL case, history and physical climate regions of the Old World, examination include skin lesions, local or Leishmania donovani infections are generalized lymphadenomegaly, emaciation, restricted to the (sub-) tropics of Asia and

evaluating the clinical progress of infected animals and may also contribute to the understanding of CanL pathogenesis. One the most remarkable characteristics of CanL-associated hematological disorders is anemia, characterized as non-regenerative normocytic or normochromic. The leucocyte Bone marrow. Macrophages, which largely replace immature erythrpoietic elements, contain alteration in the numerous 2-3um amastigotes with a central nucleus and a rod-shaped kinetoplast. (HE, 400X) blood of symptomatic dogs includes leucopenia characterized by mono- cachexia, splenomegaly, anorexia or 1 increased appetite, lethargy, temporal cytopenia, lymphopenia, and eosinopenia. muscle atrophy, exercise intolerance, The pathogenesis of hematological changes polyuria/polydipsia, ocular lesions, in both red and white blood cells is often epistaxis, onychogryphosis, lameness, related to bone marrow disorders associated vomiting, and diarrhea.4 Serum with diminished erythropoiesis due to biochemistry findings in dogs with clinical intense bone marrow parasitism. In addition, leishmaniosis include, most commonly, anemia can be related to reduced plasma serum hypoproteinemia with hyper- iron due to abnormal iron retention by globulinemia and hypoalbuminemia, re- macrophages and increased levels of sulting in a decreased albumin/globulin hepcidin, typical of anemia of chronic ratio. Mild increases of liver enzyme diseases. Anemia could also be related to activities are frequent; however, grossly increased hemolysis in enlarged spleen and elevated liver enzyme activities, severe liver associated with inflammatory response to L. infantum and to decreased production azotemia, or both, are found in only a 7 minority of dogs with leishmaniosis. of erythropoietin by damaged kidneys. Proteinuria and some renal abnormalities develop in most dogs with this disease.1 Severely affected dogs are usually cachectic and suffer from muscle atrophy. The skin Hematological parameters and the serum and hemolymphatic organs are primarily biochemical profile in L. infantum-infected affected. Generalized lymphadenomegaly dogs have limited diagnostic value. and splenomegaly are usually present. However, they can be useful biomarkers for Hepatomegaly may be present, but is less

14 common. Small nodular foci of infla- touch prep stained (Wright-Giemsa) slides mmation may develop in various organs, or in cultures of tissue aspirates or biopsy including the skin and the kidneys. Mucosal specimens of the spleen, liver, bone marrow, ulcerations in the nasal cavity, stomach, or lymph nodes.1 Immunohistochemistry is intestine, and colon are occasionally a useful tool that significantly increases the observed. The typical histopathologic sensitivity of histopathology for detecting finding in the majority of affected tissues is amastigotes, and it is a genus-specific assay an inflammatory reaction associated with to confirm the diagnosis.12 macrophages in the presence or absence of Leishmania amastigotes. Amastigote JPC Diagnosis: 1. Spleen, red pulp: numbers may vary from very few organisms Splenitis, histiocytic and plasmacytic, within macrophages to large numbers in diffuse, marked with numerous intracellular rarer events. Lymphoplasmacytic infla- amastigotes. mmation is also common in dogs with leishmaniasis.1 Additionally, renal disease 2. Spleen: Reticuloendothelial hyperplasia due to glomerulonephritis and interstitial with erythrophagocytosis and hemo- nephritis is commonly associated with CanL siderosis. due to Leishmania infantum. 3. Spleen: Extramedullary hematopoiesis.

Diagnosis of canine leishmaniasis is based 4. Bone marrow: Myelitis, histiocytic and on the presence of clinical signs together plasmacytic, diffuse, marked with numerous with positive specific antibody assay. intracellular amastigotes. Infection can be confirmed by dem- onstration of the parasites (amastigotes) on Conference Comment: Leishmania sp. organisms primarily infect the monocyte-

15 macrophage system and the visceral description above. Conference participants manifestation bears many similarities to were struck by the marked degree of histoplasmosis. Leishmania amastigotes are effacement of both the splenic red pulp and able to survive and reproduce in macrophage bone marrow. phagolysosomes due in part to an increase in pH, and macrophage destruction occurs We thank the contributor for providing secondary to proliferation of the protozoa.14 clinical pathology data with the submission, The immune response includes both T which greatly enhances the teaching value of helper 1 (Th1) and T helper 2 (Th2) the case. Participants discussed the third mediated mechanisms and tissue damage hematological evaluation and whether the occurs via a variety of methods including anemia is regenerative or nonregenerative, granulomatous inflammation, immune but without a reticulocyte count it is difficult complex deposition and the formation of to determine with certainty. The presence of autoantibodies. Inefficient killing of the nucleated red blood cells (metarubricytosis) organism by macrophages leads to the can be indicative of regeneration with various immune responses, eventually concurrent reticulocytosis, but nucleated red resulting in damage to a variety of tissues blood cells can also be present in other and an array of clinical manifestations conditions such as bone marrow damage discussed above.2 Resistance to infection is from inflammation or necrosis, lead based on a strong Th1 response, whereas a poisoning and due to splenectomy among strong Th2 response can be deleterious5 due other things. A normal MCV would support to immune complex deposition, particularly a nonregenerative anemia; additionally, the in renal glomeruli. The level of parasite pancytopenia and histologic changes in the burden also appears to play a role in the bone marrow also provide support for a immune response effectiveness against the nonregenerative anemia. Elevated ALT and invading organisms. In dogs with a high AST are indicative of mild hepatocellular parasite load CD8+ T Cells are less effective damage, which correlates with the at lysing infected macrophages. Genetic histopathologic description mentioned above susceptibility also plays a role in outcome of by the contributor in the tissues not infection with certain breeds such as the submitted. The total protein is normal but a German Shepherd, cocker spaniel, and boxer decrease in albumin and increase in globulin being more susceptible.4 The most common is likely present in this case, based on the clinical manifestations of disease include degree of infection and ongoing infla- skin, renal and/or ocular disease as well as mmation, which is a common pattern in epistaxis, and the disease is broadly divided dogs afflicted with leishmaniasis, as into cutaneous, mucocutaneous and visceral mentioned above by the contributor. forms. Enlargement of the spleen is very Contributing Institution: common and nearly always occurs in cases Veterinary School. Universidade Federal de of visceral leishmaniasis.14 While leish- Minas Gerais maniasis is more common in Mediterranean www.vet.ufmg.br countries and South America, endemic foci are also present in North America including in Texas, Oklahoma, Ohio and Michigan.5

The conference histologic description was aligned very closely with the contributor’s

References: Veterinária, Universidade Federal de Minas Gerais, 2010. 1. Baneth G, Solano-Gallego L. Leishmaniasis. In: Greene CE, ed. Infectious 9. Ready PD. Epidemiology of visceral diseases of the dog and cat. 4th ed. leishmaniasis. Clin Epidemiol. 2014; 6:147- Philadelphia, US: Elsevier Saunders; 154. 2012:735-748. 10. Romero GAS, Boelaert M. Control of 2. Cooper BJ, Valentine BA. Muscle and visceral Leishmaniasis in Latin America – A Tendon. In: Maxie MG, ed. Jubb, Kennedy, systematic review. Plos Negl Trop Dis. and Palmer's Pathology of Domestic 2010;4(1):1-17. Animals. 6th ed. Vol 1. St. Louis, MO: Elsevier; 2015:240. 11. Solano-Gallego L, Miró G, Koutinas A, et al. LeishVet guidelines for practical 3. Diniz SA, Silva FL, Carvalho Neta AV, management of canine leishmaniosis. et al. Animal reservoirs for visceral Parasite Vect. 2011;4:86. leishmaniasis in densely populated urban areas. J Infect Dev Ctries. 2008; 2:24-33. 12. Tafuri WL, Santos RL, Arantes MRE, et al. An alternative immunohistochemical 4. Koutinas AF, Koutinas CK. Pathologic method for detecting Leishmania mechanism underlying the clinical findings amastigotes in paraffin-embedded canine in canine Leishmaniosis due to Leishmania tissues. J Immunol Methods. 2004; 292:17- infantum/chagasi. Vet Pathol. 23. 2014;51(2)527-538. 13. Turchetti AP, Souza TD, Paixão TA, 5. Mauldin EA, Peters-Kennedy J. Santos RL. Sexual and vertical transmission Integumentary system. In: Maxie MG, ed. of visceral leishmaniasis. J Infect Dev Jubb, Kennedy, and Palmer's Pathology of Ctries. 2014; 8:403-407. Domestic Animals. 6th ed. Vol 1. St. Louis, MO: Elsevier; 2015:663. 14. Valli VEO, Kiupel M, Bienzle D. Hematopoietic System. In: Maxie MG, ed. 6. Ministério da Saúde. Manual de Jubb, Kennedy, and Palmer's Pathology of vigilância e controle da leishmaniose Domestic Animals. 6th ed. Vol 3. St. Louis, visceral. Brasília, BR: Editora MS; 2006. MO: Elsevier; 2015:1 7. Nicolato RC, Abreu RT, Roatt BM, et al. Clinical forms of canine visceral Leishamniasis in naturally Leishmania infantum-infected dogs and related myelogram and hemogram changes. Plos

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8. Papa DN. Perfil epidemiologico da Leishmaniose Visceral em cães diagnosticasdos no Laboratório da Escola de Veterinária da Universidade Federal de Minas Gerais Belo Horizonte 2004 a 2008. Master’s degree dissertation, Escola de

CASE IV: 21966 (JPC 4066536). From the left mandibular lymph node, similar morphologic cells were mainly Signalment: 10-year-old spayed female acquired, and several small lymphocytes, Miniature Dachshund dog, (Canis plasma cells, and eosinophils were noted. familiaris) On the smear of right mandibular lymph node, lymphoid cells, predominantly small History: The dog was presented with a mature lymphocytes with some lymph- gingival mass on right maxilla, which had oblasts and plasma cells were seen. been enlarging for 2 to 3 weeks. Both right Scattered melanophages were also observed. and left mandibular lymph nodes were enlarged by palpation. Thoracic CT scan Contributor’s Interpretation: revealed no metastasis in the lung. 1. Gingival mass: Malignant melanoma with metastasis to the left mandibular lymph node Gross Pathology: The mass measured 3.7 was suspected. x 2.4 x 1.1 cm and was partly red and black. 2. Right mandibular lymph node: Reactive Laboratory Results: None lymph node.

Cytologic description: Fine-needle asp- Histologic result: The mass was partly iration of the mass was performed. The resected for histopathology. On histologic smear was highly cellular and consisted of examination, the neoplastic cells invaded monomorphic cells, most of which are in almost all of the mass and were arranged in clusters. The cells had a round nucleus in nests supported by a fine fibrovascular pale abundant cytoplasm. The cytoplasm stroma. The neoplastic cells had clear was mild to moderately vacuolated and abundant cytoplasm and round to oval small black pigment noted in a few cells. nucleus. The cytoplasm was vacuolated and Cells showed mild to moderate anisocytosis small amount of brown pigment were and anisokaryosis, and mitotic figures were observed in several cells. A few mitotic rarely seen. figures are noted. Both left and right mandibular lymph nodes were removed and submitted for the histopathology. Neoplastic cells infiltrated both lymph nodes, and especially in the left lymph node, they spread across a wide area.

Contributor’s Comment: Tumors of melanocytic origin are relatively common in the dog. The malignancy of melanoma greatly depends on anatomic location, and oral melanoma is highly aggressive. Cytologic morphology of melanoma is various, showing the feature of epithelial cells, mesenchymal cells or discrete round Fine needle aspirate, oral mass, dog. A densely cellular, fine needle aspirate was submitted digitally for cells. Balloon cell melanoma, which is a rare examination. (Wright’s-Giemsa, 4X). variant of melanocytic tumor, has been

18 reported in human, dog and cat.1, 3,8,9,10,11 in the oral cavity. A search for melanin was Microscopically, the neoplastic cells are largely unrewarding, particularly with the amelanotic and cytoplasm is vacuolated. digital slide only being scanned to 20X, but Sebaceous carcinoma, liposarcoma, other small amounts are seen in images of the clear cell neoplasm and granulomatous histologic section provided by the con- inflammation are listed as differentials. In tributor. Aside from the differential dia- this case, sebaceous carcinoma and lipo- gnoses listed above, conference participants sarcoma were unlikely due to the location. remarked the cells resemble hepatocytes in The malignant neoplasia was more sus- animals with steroid hepatopathy, sug- picious than inflammatory disease because gesting glycogen or another similar substrate of the morphologic atypia. Due to the small may be present within the cytoplasm. amount of granules that can be melanin pigments, melanoma was the primary rule- Differential diagnoses discussed include out; immunohistochemistry such as Melan- balloon cell melanoma, rhabdomyoma, A is helpful for further diagnosis. granular cell tumor and oncocytoma. A definitive diagnosis is not possible without JPC Diagnosis: Fine needle aspirate, oral the use of immunohistochemistry, which mass: Malignant neoplasm; differentials was not performed by the contributor. include amelanotic melanoma (balloon cell), rhabdomyoma, granular cell tumor, and Balloon cell melanoma is an uncommon oncocytoma. variant of melanoma most commonly described in the skin in both domestic Conference Comment: The neoplastic cells animals and humans, and in cats is often are round to spindled and arranged localized to the head. The cells are individually and in small aggregates; the described as epithelioid, large and round, cytoplasm is amphophilic and indistinctly with abundant finely vacuolated cytoplasm vacuolated and a low to moderate subset of that generally lacks pigment.6,11 Glycogen cells contain a pale, granular eosinophilic (PAS positive) and lipid rich variants have material in the cytoplasm, the origin of been described, primarily in humans. which is unclear. The cells have a single, Although the balloon cells may contain central, round to oval nucleus with finely substances other than glycogen and other stippled chromatin, and 1-2 variably distinct similar appearing non-melanocytic tumors nucleoli. Anisocytosis is mild and may contain glycogen,11 warranting a anisokaryosis is moderate, and the N:C is cautious interpretation of cytoplasmic low; rare mitoses are observed. Other features. The background of glycogen rich salient features include nuclear molding, a neoplasms is occasionally described as central fold in the nuclear membrane of few “tigroid,” referring to a vague tiger striping cells, and rare multinucleate cells. Features pattern imparted by background con- of malignancy are not striking in this stituents.4 Ultrastructural findings in balloon sample, but the anisokaryosis, nuclear cell melanomas indicate the cytoplasmic molding and presence of mitoses warrants a vacuoles represent enlarged melanosomes. malignant interpretation. Many conference In addition to Melan A, balloon cell participants commented melanomas are us- melanomas may stain for S-100 and neuron- ually more aggressive looking, particularly specific enolase.1,11

Fine needle aspirate, oral mass, dog. Neoplastic cells are polygonal with distinct cell borders, abundant vacuolated amphophilic cytoplasm with adherent pink fibrillar matrix. Nuclei exhibit ropy chromatin with mild anisokaryosis, and binucleated cells are frequent. (HE, 120X) Rhabdomyomas, oncocytomas and granular cavity and have been conjectured to be of cell tumors have a similar cytologic app- Schwann cell origin. Rhabdomyomas and earance with eosinophilic granular material oncocytomas have benign behavior, and the in the cytoplasm, comparable to what is seen behavior of granular cell tumors is not well in a subset of cells in this case. The similar defined. Both rhadomyoma and oncocytoma cytoplasmic appearance in oncocytomas and have large pale cells with granular or foamy rhabdomyomas is related to the presence of cytoplasm. The nucleus of oncocytoma is large numbers of mitochondria, and in central, while the nucleus of rhadomyoma granular cell tumors is postulated to be due may be central or peripheral and both have to metabolic abnormalities and accumulation finely clumped chromatin and a single of lysosomes.5 Rhabomyomas are benign indistinct nucleolus. Multinucleate cells tumors of striated muscle and may occur in may be seen in rhabdomyoma. The in- the larynx and tongue of domestic animals. distinct nucleolus is a feature which may Cells may have abundant cytoplasmic help distinguish them from balloon cell glycogen, suggested by PAS positivity.7 melanoma. Granular cell tumors are variably Oncocytomas are epithelial or neur- sized but typically have abundant granular oendocrine origin neoplasms which arise eosinophilic cytoplasm and an eccentric from oncocytes or oxyphil cells, and are nucleus. Pleomorphism may be moderate in described in the larynx of young dogs. The each of the three tumors. Additional exact origin of granular cell tumors is diagnostics which may help differentiate the unclear but they primarily occur in the oral tumors include desmin positivity in

20 rhabdomyoma, cytokeratin positivity in 7. Hendrick MJ, Mahaffey EA, Moore FM, oncocytoma and PAS positivity (diastase Vos JH, Walder EJ. Histological class- resistance) in granular cell tumor.2 ification of mesenchymal tumors of the skin and soft tissues of domestic animals. 2nd series. Washington D.C.: Armed Forces Institute of Pathology. 1998:21. Contributing Institution: Laboratory of Comparative Pathology and 8. Kao GF, Helwig EB, Graham JH. Balloon Department of Diagnostic Pathology, cell malignant melanoma of the skin. A Graduate School of Veterinary Medicine, clinicopathologic study of 34 cases with Hokkaido University, Japan histochemical, immunohistochemical, and http://www.vetmed.hokudai.ac.jp/ ultrastructural observations. Cancer. 1992; 69:2942-2952. References: 9. Mowat A, Reid R, Mackie R. Balloon cell 1. Blanchard TW, Bryant NJ, Mense MG. metastatic melanoma: an important diff- Balloon cell melanoma in three dogs: a erential in the diagnosis of clear cell tumors. histopathological, immunohistochemical and Histopathology. 1994; 24:469-472. ultrastructural study. J Comp Pathol. 2001; 125:254-261. 10. van der Linde-Sipman JS, de Wit MM, van Garderen E, Molenbeek RF, et al. 2. Burkhard MJ. Respiratory Tract. In: Cutaneous malignant melanomas in 57 cats: Raskin RE, Meyer DJ, eds. Canine and identification of (amelanotic) signet-ring and Feline Cytology. 3rd ed. St. Louis, MO: balloon cell types and verification of their Elsevier; 2016:155-156. origin by immunohistochemistry, electron microscopy, and in situ hybridization. Vet 3. Cangul IT, van Garderen E, van der Pathol. 1997; 34:31-38. Linde-Sipman JS, van den Ingh TS, Schalken JA. Canine balloon and signet-ring 11. Wilkerson MJ, Dolce K, DeBey BM, cell melanomas: a histological and imm- Heeb H, et al. Metastatic Balloon Cell unohistochemical characterization. J Comp Melanoma in a Dog. Vet Clin Pathol. 2003; Pathol. 2001; 125:166-173. 32(1):31-6. 4. DeMay RM. The Art and Science of Cytopathology. Vol 2. 2nd ed. Hong Kong: American Society for Clinical Pathology; 2012:592.

5. Dunbar MD, Ginn P, Winter M, Miller KB, Craft W. Laryngeal rhabdomyoma in a dog. Vet Clin Pathol. 2012; 41(4):590-593. 6. Goldschmidt MH, Dunstan RW, Stannard AA, Tscharner CV, et al. Histological classification of epithelial and melanocytic tumors of the skin of domestic animals. Vol III. 2nd series. Washington D.C.: Armed Forces Institute of Pathology. 1998:38-39.