Pathology in Practice

Ioannis L. Oikonomidis From Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, Scotland

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Pierre Boye The Hospital for Small Animals, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, Scotland

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Silke Salavati Schmitz The Hospital for Small Animals, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, Scotland

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Luca Aresu Department of Veterinary Science, University of Turin, Turin, 10095, Italy.

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Elspeth Milne From Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, Scotland

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Chiara Piccinelli From Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, Scotland

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History

A 3.5-year-old 18.7-kg sexually intact female Chinese Shar-Pei was referred for further investigation of azotemia and proteinuria. The dog had a 2-year history of recurrent episodes of lethargy, pyrexia, lameness of the hind limbs, and swollen, hot tarsal joints. These clinical signs were attenuated with administration of NSAIDs. The dog's vaccination status was current. The dog received regular deworming and had no history of travel outside Scotland.

Clinical and Clinicopathologic Findings

Results of a complete physical examination were unremarkable, although systemic blood pressure measured by the Doppler ultrasonographic method was mildly high (150 mm Hg; reference interval, < 150 mm Hg). A CBCa revealed mild, nonregenerative anemia (Hct, 34.2% [reference interval, 39% to 55%]; reticulocyte count, 23.6 × 109/L [reference interval, < 60 × 109/L]). Moderate acanthocytosis and mild schistocytosis were noted on blood smear examination. A comprehensive serum biochemical profileb revealed moderate azotemia (urea concentration, 15.3 mmol/L [reference interval, 1.7 to 7.4 mmol/L]; creatinine concentration, 212 μmol/L [reference interval, 22 to 115 μmol/L]), mild hypoalbuminemia (22.6 g/L; reference interval, 26 to 35 g/L), mild hypercholesterolemia (10.3 mmol/L; reference interval, 3.8 to 7.0 mmol/L), and mild hyperglycemia (6.1 mmol/L; reference interval, 3 to 5 mmol/L). Routine coagulation panel was unremarkable. Urinalysis revealed low urine specific gravity (1.015) and high urine pH (8). Urine protein-to-creatinine ratiob was markedly high (8.0; reference interval, < 0.2). Urine sediment was unremarkable, and no bacteria were recovered from culture of a urine sample. The dog was seronegative for Leptospira interrogans serovars Bratislava, Canicola, Copenhageni, and Icterohemorrhagiae (determined by microscopic agglutination test with cutoff titer of 1:100), and Leptospira spp were not detected in a urine sample by PCR assay.

Thoracic radiographic views were unremarkable. Abdominal ultrasonography revealed a moderately enlarged liver with mildly heterogeneous echotexture, gallbladder sludge, mildly and diffusely stippled splenic echotexture, bilateral mild diffuse adrenomegaly, and mild colonic wall thickening.

Ultrasound-guided fine-needle aspirate specimens of the spleen and liver were obtained. Splenic aspirate specimens had variably sized aggregates of reticular cells and a mixed population of lymphocytes (mostly small) that were usually embedded in abundant, smooth, purple to eosinophilic, extracellular material arranged in irregular clumps (Figure 1). In the hepatic aspirate specimens, there were variably sized clusters of hepatocytes that often contained moderate numbers of discrete to smudged cytoplasmic vacuoles. A moderate amount of the same eosinophilic extracellular material found in the splenic aspirate specimens was frequently observed among the hepatocytes.

Figure 1
Figure 1

Photomicrographs of ultrasound-guided fine-needle aspirate specimens obtained from the spleen (A) and liver (B) of a 3.5-year-old Chinese Shar-Pei that was referred for further investigation of azotemia and proteinuria. A—Moderate numbers of erythrocytes are present in the background. A large amount of purple extra-cellular material is observed in the periphery of an aggregate of reticular cells. A few small and intermediate lymphocytes and nondegenerate neutrophils are found. May-Grünwald Giemsa stain; bar = 50 μm. B—A few small clusters of hepatocytes that often contain moderate numbers of small discrete cytoplasmic vacuoles (lipids). A moderate amount of the same eosinophilic extracellular material is observed among the hepatocytes. May-Grünwald Giemsa stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 259, 5; 10.2460/javma.259.5.485

Cytologic Findings

The extracellular material observed in the splenic and hepatic aspirate smears was suspected to be amyloid. To further investigate the origin of the material, 1 hepatic and 2 splenic aspirate smears were destained and restained with Congo red stain. When observed by light microscopy, the smooth extracellular material in the aspirate specimens appeared red (Figure 2). When examined under polarized light, the Congophilic material had apple-green birefringence in the thicker areas of the splenic aspirate specimen and in a few areas of the liver aspirate (data not shown). Those findings confirmed the presence of amyloid deposition in both organs.

Figure 2
Figure 2

Photomicrographs of ultrasound-guided fine-needle aspirate specimens from the dog's spleen after Congo red staining. A—In this section, abundant Congophilic (red) material is observed. Congo red stain; bar = 200 μm. B—When the same field as shown in panel A is viewed under polarized light, the Congophilic material has apple-green birefringence in the thicker areas. Congo red stain; bar = 200 μm.

Citation: Journal of the American Veterinary Medical Association 259, 5; 10.2460/javma.259.5.485

Histopathologic, Immunofluorescence, and Ultrastructural Findings

In renal tissue sections, 2 of 12 glomeruli available for examination were globally sclerotic. All glomeruli had moderate mesangial expansion by amorphous, extra-cellular, eosinophilic, and Congophilic material (Figure 3). The material was pale pink when stained with periodic acid–Schiff stain and mottled blue and pale peach when stained with Masson trichrome stain; the material did not stain with Jones methenamine silver stain. Segmentally, glomerular capillary walls were also expanded by amyloid, often resulting in capillary wall compression and ischemic damage characterized by sclerosis. Amyloid was not detected in the interstitial compartment, including vessels. Diffuse tubular epithelial degeneration associated with multifocal necrosis and tubular ectasia attributable to large protein casts were also present. Interstitial fibrosis was mild and patchy, and there were a few multi-focal atrophic tubules.

Figure 3
Figure 3

Photomicrographs and transmission electron micrographs of sections of renal tissue. A—For the glomerulus in this view, the mesangium is severely expanded by eosinophilic amorphous material. There is effacement of peripheral capillary loops by amyloid. H&E stain; bar = 50 μm. B—Amyloid deposition in a glomerulus is Congophilic (stained orange to peach). Congo red stain; bar = 30 μm. C—With transmission electron microscopy, amyloid fibrils appear densely packed and organized in the glomerular basement membrane (arrows). Uranyl acetate and lead citrate stain; bar = 2 μm. D—At higher magnification, the haphazard arrangement of the amyloid fibrils is evident. Uranyl acetate and lead citrate stain; bar = 2 μm.

Citation: Journal of the American Veterinary Medical Association 259, 5; 10.2460/javma.259.5.485

One renal biopsy specimen was evaluated with immunofluorescence tests and was negative for IgG, IgA, IgM, and complement component C3. These findings were not consistent with underlying immune-mediated glomerulonephritis.

Ultrastructural evaluation of a section of renal tissue by transmission electron microscopy revealed glomeruli with diffuse areas with visceral epithelial cell foot-process fusion, and the filtration membrane and mesangium had confluent areas of thickening associated with aggregates of fine, fibrillary, nonbranching structures (measuring 8 to 15 nm in width), morphologically consistent with amyloid (Figure 3).

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: suspected splenic amyloidosis, suspected hepatic amyloidosis, mild to moderate vacuolar hepatopathy, moderate to severe glomerular amyloidosis, and marked diffuse acute tubular epithelial degeneration associated with multifocal necrosis and frequent tubular ectasia.

Case summary: systemic amyloidosis in a dog with familial Shar-Pei fever.

Comments

Amyloidosis is characterized by the extracellular deposition of fibrillar proteins in tissues.1 These insoluble fibrils are produced by an aggregation-prone protein and can cause tissue damage and loss of function.1 In human medicine, more than 20 amyloid precursors have been identified, at least 8 of which have also been reported in the veterinary medical literature.2 Systemic amyloidosis can be clinically classified as primary (amyloid light chain amyloidosis) or secondary (or reactive) amyloid-associated amyloidosis; heredofamilial amyloidosis is a collective term for a heterogeneous group of inherited syndromes.1

In domestic animals, immunoglobulin light chain amyloidosis is a rare entity associated with overproduction of monoclonal immunoglobulin light chains by neoplastic plasma cells.2 In veterinary medicine, amyloid-associated amyloidosis is the most common form of systemic amyloidosis and is associated with deposition of amyloid A, an N-terminal fragment of the acute-phase protein serum amyloid A. Amyloid-associated amyloidosis is closely associated with chronic inflammatory diseases or neoplasia.2 Familial amyloidosis in Shar-Peis and in Siamese and Abyssinian cats has been reported, but it is still considered a type of amyloid-associated amyloidosis for which those breeds have a genetic predisposition.2,3

Familial amyloidosis of Shar-Peis appears to be linked to familial Shar-Pei fever (or Shar-Pei autoinflammatory disease), a disease that is characterized by short, recurrent episodes of pyrexia and localized inflammation, usually involving the tarsal joints. It is postulated that the episodes of inflammation lead to a subclinical, chronic autoinflammatory state that can predispose Shar-Peis to the development of amyloid-associated amyloidosis.4

In dogs (including Shar-Peis), amyloidosis primarily affects the kidneys but it can also affect various other organs.5,6,7 Notwithstanding, amyloid is rarely detected in cytologic specimens and can be easily overlooked or misinterpreted as stroma, basement membrane–associated material, osteoid, colloid, or inspissated mucin, depending on the examined site.8 Congo red stain is most frequently used to confirm amyloid deposition in both cytologic and histologic specimens.9 When viewed under polarized light, amyloid has apple-green birefringence because of the parallel alignment of the amyloid fibrils and dye molecules.8 However, this characteristic is dependent on section thickness (ideally 8 to 10 μm); in thinner sections (eg, in some areas of cytologic preparations), the characteristic apple-green birefringence may not be evident.10 Thioflavin-T staining, transmission electron microscopy, and immunohistochemical analysis (performed with an antibody against an amyloid precursor) can also be used to confirm amyloidosis; however, such procedures are not usually required for a definitive diagnosis if Congo red staining has been successfully performed on cytologic or histologic specimens.9

In contrast to the first report7 regarding renal amyloidosis in Shar-Peis, which favored medullary rather than glomerular deposition of amyloid, diffuse glomerular amyloidosis was a consistent histopathologic finding in a subsequent retrospective study.5 In the case described in the present report, the moderate to severe glomerular amyloidosis could explain the dog's moderate renal azotemia, marked proteinuria with hypoalbuminemia, and mild nonregenerative anemia (possibly attributable to a combination of diminished erythropoietin production by the affected kidneys and anemia of inflammatory disease), as well as the presence of acanthocytes and schistocytes.11 The dog also had mild hypercholesterolemia, which is a relatively common finding in dogs with renal amyloidosis5,6,7; hypercholesterolemia is postulated to be associated with a high serum concentration of amyloid A, which acts as an apolipoprotein of high-density lipoproteins and a transporter of cholesterol in the circulation.12

In cytologic specimens, amyloid deposits may be difficult to recognize and amyloidosis may be misdiagnosed. The examination of Congo red–stained cytologic or histologic preparations under polarized light is recommended to confirm the diagnosis of amyloidosis, although transmission electron microscopy can also be used for this purpose.

Footnotes

a.

ADVIA 2120, Siemens Healthcare Diagnostics Ltd, Malvern, Pa.

b.

AU480, Beckman Coulter, Brea, Calif.

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