Pathology in Practice

Adi Wasserkrug Naor Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Melinda J. Wilkerson Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Mandy Meindel Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Mark Morton Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Lisa M. Pohlman Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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History

A 2.5-year-old 40.8-kg (89.8-lb) neutered male German Shepherd Dog was evaluated because of swelling of the limbs, tongue, and periocular region bilaterally of 1 month's duration. Several days prior to the evaluation, the owners noticed that the dog seemed to have signs of pain in multiple joints. In addition, the dog had apparently painful ulcers on the left elbow, right forepaw, and lingual area. The dog's appetite declined 2 days prior to evaluation.

Clinical and Clinicopathologic Findings

On physical examination, the dog was febrile (39.9°C [103.8°F]) and tachycardic (heart rate, 150 beats/min), and had a respiratory rate of 48 breaths/min. The dog was recumbent and unable to stand. The dog had multiple swollen joints. Signs of pain were elicited when the dog's joints were palpated; pitting edema of the limbs was evident. On the dog's left elbow, there was a large draining wound (Figure 1). There was a similar lesion on its right forepaw. The prescapular and popliteal lymph nodes were enlarged. Oral examination revealed sublingual swelling and lingual ulcerations.

Figure 1—
Figure 1—

Photograph of a large draining wound (arrow) on the left elbow of a 2.5-year-old German Shepherd Dog that was evaluated because of swelling of the limbs, tongue, and periocular region bilaterally of 1 month's duration.

Citation: Journal of the American Veterinary Medical Association 250, 6; 10.2460/javma.250.6.627

A CBC revealed an acute inflammatory leukogram (possibly with a stress leukogram component) characterized by leukocytosis (38.6 × 103 WBCs/μL; reference interval, 6 × 103 WBCs/μL to 17 × 103 WBCs/μL) as a result of neutrophilia (35.5 × 103 neutrophils/μL; reference interval, 3 × 103 neutrophils/μL to 11.5 × 103 neutrophils/μL) with a left shift (3.5 × 103 bands/μL; reference interval, 0 to 0.3 × 103 bands/μL), lymphocytopenia (0.8 × 103 lymphocytes/μL; reference interval, 1.5 × 103 lymphocytes/μL to 5 × 103 lymphocytes/μL), and monocytosis (1.9 × 103 monocytes/μL; reference interval, 0.1 × 103 monocytes/μL to 0.8 × 103 monocytes/μL).

Serum biochemical analysis revealed mild hyperglycemia (127 mg/dL; reference interval, 73 to 113 mg/dL) and mild hyperglobulinemia (3.3 g/dL; reference interval, 1.3 to 3.2 g/dL), with no additional remarkable findings. A urine sample was collected by cystocentesis; urine specific gravity was 1.043. Urinalysis (by dipstick) revealed a pH of 7, 2+ bilirubin, 3+ heme, and 3+ protein. A sulfosalicylic acid assay indicated that the urine protein concentration was 100 mg/dL; dogs with concentrated urine without evidence of urinary tract disease can have maximum of 1+ protein reaction in a dipstick analysis, which is equivalent to 30 mg of protein/dL.1 The only abnormality found on microscopic examination of urine sediment was > 200 erythrocytes/hpf. Microbial culture of a urine sample yielded no growth.

Fine-needle aspiration of synovial fluid from the dog's left elbow joint was performed. On gross examination, the synovial fluid sample was cloudy and red; after centrifugation, the fluid became clear and colorless. The total nucleated cell count was 4 × 103 cells/μL. Synovial fluid samples from clinically normal dogs typically contain < 3 × 103 nucleated cells/μL.2 The PCV was < 3%. The protein concentration was not measured because of insufficient sample volume.

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Cytologic Findings

The synovial fluid sample was submitted for clinicopathologic evaluation. The direct preparations had low nucleated cellularity, occasional erythrocytes, and a pale background. As part of the regular sample preparation, hyaluronidase was added to the synovial fluid prior to sample analysis. Because hyaluronidase breaks down hyaluronic acid (the substance responsible for the viscosity of joint fluid), there was no evidence of windrowing of RBCs. Nucleated cells were primarily nondegenerate to mildly degenerate neutrophils (approx 70%) with small lymphocytes (approx 15%) and large mononuclear cells (approx 15%). Of the neutrophils, approximately 30% contained a pink cytoplasmic inclusion. The pink inclusions were round, ranged in diameter from 2 to 4 μm, had a homogeneous smooth appearance, and frequently caused peripheralization of the nucleus. The cytomorphological features of these inclusions were consistent with nuclear material (Figure 2), indicative of lupus erythematosus (LE) cells. No microorganisms were identified.

Figure 2—
Figure 2—

Photomicrograph of a cytologic preparation of a sample of synovial fluid collected from the left elbow joint of the dog in Figure 1. In the center of the image, there is a lupus erythematoses cell. The nucleus of the neutrophil is pushed to the cell border, and the cell contains 1 round (approx 4.5-μm-diameter) pink inclusion body (arrow). Erythrocytes are present in the background. Modified Wright stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 250, 6; 10.2460/javma.250.6.627

Additional Clinicopathologic Findings

An anti-nuclear antibody (ANA) test was performed by incubating a sample of the dog's serum on rat liver sections.a After 30 minutes, the slide was washed 3 times with PBS solution, and then incubated with a fluorescein isothiocyanate-conjugated rabbit anti-dog IgG antibody.b The section was washed again and counterstained with Evans blue stain.c The result of the indirect fluorescent antibody test was positive with homogeneous nuclear staining, with all dilutions of dog serum from 1:80 to 1:5,120 (Figure 3).

Figure 3—
Figure 3—

Photomicrograph of a section from a rat liver incubated with a sample of serum from the dog in Figure 1 in an anti-nuclear antibody indirect immunofluorescence assay. After 30 minutes, the slide was washed 3 times with PBS solution, and then incubated with a fluorescein isothiocyanate-conjugated rabbit anti-dog IgG antibody. The section was washed again and counterstained with Evans blue stain. At a serum dilution of 1:640, the result of the indirect fluorescent antibody test was positive with homogeneous nuclear staining. Bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 250, 6; 10.2460/javma.250.6.627

Clinicopathologic Diagnosis and Case Summary

Systemic lupus erythematosus (SLE) in a dog.

Comments

Systemic lupus erythematosus is a rare prototype of multiorgan autoimmune disease.3 The pathogenesis of SLE is multifactorial and is associated with genetic and environmental factors as well as abnormalities of both the innate and the adaptive immune system.4 Systemic lupus erythematosus develops most commonly in middle-aged, medium- to large-breed dogs of either sex. The most commonly reported clinical signs include polyarthritis, glomerulonephritis, skin lesions, and hemolytic anemia. There is a notable seasonal pattern for SLE with most cases occurring in the summer and fall.5,6

Lupus erythematosus cells are neutrophilic phagocytes that contain intracytoplasmic hematoxylin bodies of nuclear material. The hematoxylin bodies are believed to be formed by the opsonization of nuclear material, which leads to neutrophil engulfment, thereby creating LE cells.7 In people with SLE, LE cells have been detected in samples of pleural effusions, bone-marrow aspiration material, synovial fluid, CSF, and pericardial fluid.8,9

A diagnosis of SLE in dogs is typically made on the basis of ≥ 2 clinical signs and serum ANA titer. The ANA test detects antibodies in the patient's serum that react with nuclear antigens. Two substrates are commercially available for ANA testing including human epithelial cells (HEP)-2 and rat liver tissue. Hansson et al10 found that 95% of healthy dogs were considered negative only after a serum ANA titer dilution > 1:100 was used as the cutoff value. Smee et al11 reported that a diagnosis of SLE should be considered either when the serum ANA test is positive at serum dilutions > 1:640 or when a patient has a lower serum ANA titer (> 1:160) and at least 2 major clinical signs (ie, skin lesions, polyarthritis, hemolytic anemia, glomerulonephritis or substantial proteinuria, polymyositis, leukopenia, or thrombocytopenia). With the HEP-2 substrate, a dog can be considered positive for SLE when ANAs are detected at serum dilutions of ≥ 1:100.12 Hansson-Hamlin et al12 also suggested that different nuclear fluorescence patterns might be associated with different systemic autoimmune diseases in dogs. Dogs with homogeneous nuclear (chromosomal) staining had several major clinical signs that highly correlated with the diagnosis of SLE. However, speckled nuclear staining was more common in dogs that had restricted clinical signs including musculoskeletal disorders, fever, and fatigue. It is possible that the speckled nuclear staining pattern is evidence of SLE-related diseases, which may be associated with different immunodiffusion subgroups. The differentiation of these nuclear staining patterns is generally difficult when rat liver tissue is used as a substrate.10,12

The case described in the present report was not diagnostically challenging because of the diagnostic indicators of SLE, but is interesting because of the plethora of evidence. A diagnosis of SLE was very strongly indicated on the basis of the ANA test results alone (positive in a serum dilution of 1:5,120). In addition, the dog had major clinical signs indicative of SLE, including skin lesions and polyarthritis, and additional minor clinical signs including lymphadenomegaly and oral ulcers. A draining or ulcerated wound as a result of dermal-epidermal separation secondary to apoptosis of basal cells is common in dogs with SLE3; this dog had a large draining wound on its left elbow. Identification of LE cells in the synovial fluid sample from the dog, even in the absence of the other clinical findings, was strongly suggestive of SLE.

The prognosis for dogs with SLE is variable. The condition of some dogs might be well controlled with treatment and remission is possible, although relapse should be anticipated; however, other dogs may have no improvement with treatment, as occurred in the case described in the present report. A correlation between ANA test results and clinical behavior of the disease has been reported.13 Treatments for dogs with SLE usually involve administration of immunosuppressive doses of glucocorticoids. Adjunct cytotoxic drugs may be considered depending on the severity of the clinical signs. A combination treatment of glucocorticoids with levamisole (3 to 7 mg/kg [1.36 to 3.18 mg/lb]) given orally appears to be more effective than oral corticotherapy alone.6 Some reports indicate that dogs that did not respond to conventional treatment achieved clinical remission when they were treated with plasmapheresis and low-dose immunosuppressive drug treatment.14

The dog of the present report was treated with lactated Ringer solution with KCl (16 mEq/L) administered IV at a rate of 100 mL/h. Various antimicrobials, analgesics, famotidine, and immunosuppressive drugs were administered. After 13 days of treatment, the result of a repeated ANA test was positive (1:640) and the dog failed to have any clinical improvement. Owing to quality-of-life concerns, the owners elected euthanasia.

Footnotes

a.

Rat liver tissue for antinuclear antibody test, Medica, San Diego, Calif.

b.

AffiniPure rabbit anti-dog IgG (H+L) fluorescein isothiocyanate (FITC) conjugated, Jackson ImmunoResearch, West Grove, Pa.

c.

Evans blue (diluted to 0.003%), Acros Organics, Fisher Scientific, Houston, Tex.

d.

Unasyn, Auromedics Pharma LLC, Dayton, NJ.

e.

Baytril, Bayer, Shawnee Mission, Kan.

f.

Cuprofloxacin tablets, PACK Pharmaceuticals, Buffalo Grove, Ill.

g.

Dilaudid, West-Ward Pharmaceuticals, Eatontown, NJ.

h.

Mucosolvan, Roxane Laboratories, Columbus, Ohio.

i.

Gabapentin capsules, Sun Pharmaceutical Industries Ltd, Cranbury, NJ.

j.

Famotidine injection, West-Ward Pharmaceuticals, Eatontown, NJ.

k.

Dexamethasone-SP, Vet One, Boise, Idaho.

l.

Prednisone, Roxane Laboratories, Columbus, Ohio.

m.

Sandimmune, Novartis Pharmaceuticals, East Hanover, NJ.

References

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