Pathology in Practice

Stephanie M. ShraderDepartment of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Eric J. FishDepartment of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Mariano Mora PereiraDepartment of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Erin S. GrooverDepartment of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Peter W. ChristophersonDepartment of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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History

A 14-year-old Missouri Fox Trotter gelding was evaluated because of chronic weight loss of 1 year's duration and acute epistaxis from the left nostril that occurred 2 days prior.

Clinical and Clinicopathologic Findings

On physical examination, the horse was quiet but responsive. It weighed 515 kg (1,133 lb) and had an estimated body condition score of 4/9 with loss of muscle over the dorsum. Rectal temperature was 38.3°C (100.9°F), heart rate was 48 beats/min, and respiratory rate was 12 breaths/min. The horse's oral mucous membranes were pale pink; spontaneous mucosal bleeding occurred while assessing capillary refill time, which was < 2 seconds. Epistaxis was apparent from both nares. The mandibular lymph nodes were markedly enlarged and firm. Endoscopic evaluation of the upper airways revealed petechial hemorrhages throughout the nasal passages, pharynx, larynx, and trachea. Hematologic and serum biochemical analyses revealed multiple clinically important abnormalities (Table 1) including thrombocytopenia, leukopenia, and anemia. Ultrasonographic evaluation of the abdomen revealed segmental thickening of the small intestine and free peritoneal fluid; the spleen had mixed echogenicity. Analysis of a peritoneal fluid sample collected via abdominocentesis revealed a low nucleated cell concentration (900 WBCs/μL) as well as a low protein concentration (0.6 g/dL). Aspirate specimens of the mandibular lymph nodes and bone marrow were submitted for cytologic examination. A bone marrow core biopsy specimen was collected from the sternum and submitted for histopathologic evaluation. However, owing to the continued decline in the horse's clinical condition, it was euthanized by means of IV administration of euthanasia solution prior to finalization of the report of the bone marrow biopsy findings.

Table 1—

Hematologic and serum biochemical abnormalities detected at the initial evaluation of a 14-year-old Missouri Fox Trotter gelding that was evaluated because of chronic weight loss of 1 year's duration and acute epistaxis from the left nostril that occurred 2 days prior.

Hematologic abnormalitiesBiochemical abnormalities
VariableValueReference rangeVariableValueReference range
Hct (%)25.732–48Total protein (g/dL)5.946–8.6
Hemoglobin (g/dL)9.210–18Albumin (g/dL)2.32.7–4.1
RBC concentration (× 106/μL)4.856–12Globulin (g/dL)3.72.8–4.4
Mean corpuscular volume (fL)53.134–58Sorbitol dehydrogenase (U/L)3.40.5–10
Mean corpuscular hemoglobin concentration (g/dL)35.731–37Aspartate aminotransferase (U/L)226144–350
   γ-Glutamyltransferase (U/L)142–29
RBC distribution width (%)19.817–20Total bilirubin (mg/dL)1.280.7–3.6
Platelet concentration (× 103 platelets/μL)19100–230Creatine kinase (U/L)177103–402
Mean platelet volume (fL)7.85.8–11.5BUN (mg/dL)9.89–24
   Creatinine (mg/dL)10–2
Total leukocyte concentration1.86–12Calcium (mg/dL)10.910.5–12.9
(× 103 WBCs/μL)  Phosphorus (mg/dL)2.82.1–4.6
Neutrophil concentration0.3783–6Magnesium (mg/dL)1.61.7–2.1
(× 103 neutrophils/μL)  Glucose (mg/dL)11681–127
Band concentration (× 103 bands/μL)0.0180–0.1Bicarbonate (mmol/L)26.521–30
Total lymphocyte concentration1.1341.5–5Sodium (mmol/L)137134–150
(× 103 lymphocytes/μL)  Potassium (mmol/L)3.43.5–4.5
Monocyte concentration0.2160–0.6Chloride (mmol/L)9997–111
(× 103 monocytes/μL)  Anion gap14.99–25
Eosinophil concentration0.0180–0.6Osmolality, calculated (mOsm/kg)271276–288
(× 103 eosinophils/μL)  Iron (μg/dL)319129–173

Other morphologic findings included moderate echinocytes, slight rouleaux, and some activated or reactive lymphocytes. The manual platelet estimate was approximately 33,000 to 44,000 platelets/μL.

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

Figure 1—
Figure 1—

Photomicrograph of a cytologic preparation from the right mandibular lymph node of a 14-year-old Missouri Fox Trotter gelding that was evaluated because of chronic weight loss of 1 year's duration and acute epistaxis from the left nostril that occurred 2 days prior. Notice that these cells have scant to moderate amounts of basophilic cytoplasm with few to moderate numbers of large magenta granules, small to intermediate-sized nuclei (approx 1 to 1.5 times the diameter of an RBC), nuclear indentations, condensed chromatin, and absence of visible nucleoli. The background contains numerous cytoplasmic fragments (so-called lymphoglandular bodies). Modifed Wright stain; bar = 15 μm.

Citation: Journal of the American Veterinary Medical Association 252, 12; 10.2460/javma.252.12.1477

Cytologic Findings

Cytologic evaluation of one of the mandibular lymph node aspirates revealed a heterogeneous population of lymphocytes (approx 50% to 60% small lymphocytes, 30% intermediate lymphocytes, and approx 10% large lymphocytes). Approximately 80% to 90% of all lymphocytes contained multiple large, irregular, magenta cytoplasmic granules in 1 area of the cytoplasm (large granular lymphocyte [LGL] morphology; Figure 1). Many of the small and intermediate lymphocytes also had cytoplasmic projections (a so-called hand mirror appearance). On the basis of the findings for this lymph node, a presumptive diagnosis of lymphoid neoplasia with LGL morphology was made. The abdominal effusion was interpreted as a protein-poor transudate, but low numbers of small LGLs were also observed in this fluid. The bone marrow aspirate specimen was markedly hypocellular but contained a moderate number of small to intermediate LGLs, along with very rare hematopoietic cells. In light of the findings that supported lymphoid neoplasia, the marked pancytopenia was attributed to decreased production secondary to infiltrative neoplasia within the bone marrow. Interestingly, the horse's serum iron concentration was markedly high; given the absence of hemolysis or administration of a transfusion or exogenous iron, this abnormality was most likely secondary to ineffective erythropoiesis.

Gross and Histopathologic Findings

Necropsy revealed petechial hemorrhages scattered throughout the tracheal mucosa and pulmonary parenchyma. There was approximately 500 mL of serosanguineous pericardial effusion. All examined lymph nodes (including mandibular, tracheobronchial, splenic, hepatic, mesenteric, and inguinal lymph nodes) were markedly enlarged (up to 8 × 5 × 4 cm); on cut surface, the nodes were edematous, and the normal parenchyma was effaced by coalescing, soft, 1-to 3-mm-diameter, white to tan nodules. The spleen was subjectively enlarged with rounded margins; the capsular surface had numerous petechial hemorrhages and a cobblestone texture. The splenic parenchyma bulged on a cut surface and had a soft cobblestone texture with scattered 0.5- to 1.5-cm-diameter, soft, dark red nodules that blended imperceptibly into the surrounding parenchyma.

On histologic examination, sections of the lymph nodes were effaced by dense, coalescing, ill-defined nodules of neoplastic round cells (Figure 2). Individual neoplastic cells were approximately 10 μm in diameter with well-delineated cellular borders, scant amounts of amphophilic cytoplasm containing hypereosinophilic granules that were typically 0.5 to 1 μm in diameter, round to irregularly shaped or indented nuclei, coarsely clumped chromatin, and indistinct nucleoli (Figure 3). The intracytoplasmic granules appeared dark blue following phosphotungstic acid–hematoxylin staining, and they often clustered within the nuclear indentations. Anisocytosis and anisokaryosis were mild; there was 1 mitotic figure/10 consecutive hpf (400X). Apoptotic neoplastic cells were occasionally observed. The neoplastic cells had strong cytoplasmic immunoreactivity for CD3 (a T-cell marker) and lacked immunolabeling for Pax5 (a B-cell marker). Scattered throughout the parenchyma were small aggregates of round cells that had strong Pax5 nuclear immunolabeling (representing remnant follicular arrangements) admixed with low numbers of plasma cells. In addition to the neoplastic cellular infiltrate, medullary and subcapsular sinuses were moderately congested and contained more than normal numbers of plasma cells and histiocytes. Similar neoplastic cells expanded and effaced the portal triads, splenic white and red pulp, lamina propria and submucosa of the small and large intestines, and renal interstitium. Within the lungs, the neoplastic cells expanded perivascular, peribronchiolar, and alveolar septal interstitial spaces. Multiple sections of bone marrow were evaluated (all collected from the left femur during necropsy); all sections were composed entirely of adipose tissue, scattered small-caliber blood vessels, and a few bony spicules. In conjunction with the postmortem bone marrow samples, the antemortem core bone marrow sample was also reviewed. Sections of the latter were paucicellular with multiple, variably sized pools of marrow surrounded by histologically unremarkable trabecular bone and cartilage. The hematopoietic spaces contained abundant adipose tissue (comprising approx 70% of the overall cell population) and low numbers of spindle cells. Megakaryocytes were uncommon (typically 1 in 5 hpf [400X]). Decalcification of the sample resulted in loss of cellular detail, precluding an in-depth evaluation of cellular and nuclear morphologies and cellular maturation or estimation of the myeloid-to-erythroid cell ratio. Scattered within the marrow, however, were rare granular lymphocytes that resembled the neoplastic cells.

Figure 2—
Figure 2—

Photomicrograph of a section of the right mandibular lymph node from the horse in Figure 1. The lymph node is effaced by dense, coalescing, ill-defined nodules of neoplastic lymphocytes. H&E stain; bar = 500μm.

Citation: Journal of the American Veterinary Medical Association 252, 12; 10.2460/javma.252.12.1477

Figure 3—
Figure 3—

Photomicrographs of sections of the right mandibular lymph node from the horse in Figure 1. A—The neoplastic lymphocytes have scant amounts of amphophilic cytoplasm containing hypereosinophilic granules (arrow) and round to irregularly shaped or indented nuclei. H&E stain; bar = 40 μm. B—Following phosphotungstic acid–hematoxylin staining, the intracytoplasmic granules appear dark blue (arrow). Phosphotungstic acid–hematoxylin stain; bar = 30 μm. C—The cytoplasmic immunoreactivity with anti-CD3 antibody (brown labeling) of almost all neoplastic cells identifies them as T lymphocytes. Immunohistochemical CD3-specifc stain, diaminobenzidine chromogen, and hematoxylin counterstain; bar = 30 μm.

Citation: Journal of the American Veterinary Medical Association 252, 12; 10.2460/javma.252.12.1477

Interpretation and Case Summary

Interpretation: disseminated lymphoma (large granular lymphocyte morphology) with concurrent bone marrow hypocellularity and pancytopenia.

Case summary: disseminated T-cell lymphoma (with LGL morphology) in a horse.

Comments

Large granular lymphocyte leukemia or lymphoma in horses,1–5 dogs,6 cats,7 rats,8 and people9 has been reported and results from the neoplastic expansion of either cytotoxic T lymphocytes or natural killer cells. Both of these cell types are lymphocytes with coarse, azurophilic, cytotoxic, cytoplasmic granules. Although they have similar morphologic characteristics as revealed by cytologic evaluation and H&E staining, they can be differentiated with immunohistochemical labeling. Cytotoxic T lymphocytes have a CD3+ CD8+ CD4– immunophenotype, whereas natural killer cells have a CD3– CD16+ immunophenotype.9 The neoplastic cells in the horse of the present report were CD3 immunopositive, consistent with neoplasia of cytotoxic T-lymphocyte origin.

Although lymphoma with LGL morphology in horses has been rarely documented,1–5 the reported cases share multiple similarities. As observed in the horse of the present report, some horses have disseminated disease that affects various organs including the spleen, lymph nodes, liver, intestines, pancreas, and kidneys.1–5 An additional commonality of the reported cases with the lymphoma in this horse was the presence of neoplastic cells in peritoneal fluid.1,2,4,5 Weight loss (as observed in the horse of the present report) in addition to lethargy and diarrhea are typical clinical signs in affected horses.1–5

To the authors' knowledge, this is the first reported case of equine LGL lymphoma with concurrent bone marrow hypocellularity and pancytopenia. In a recent study10 of 203 people with T-cell granular lymphocytic leukemia, 4% of patients also had aplastic anemia. In such cases, it has been proposed that pancytopenia results from expansion of the neoplastic cytotoxic T-cell population; these cells suppress hematopoiesis via direct cellular cytotoxic effects or cytokine production. With the exception of a relative or absolute increase in LGLs in the peripheral blood or bone marrow, patients with LGL lymphoma have peripheral blood and bone marrow findings that resemble those described for people with acquired aplastic anemia.11 In the horse of the present report, epistaxis, spontaneous oral mucosal bleeding, and nasopharyngeal and laryngotracheal petechiae were likely a result of bone marrow suppression and subsequent severe thrombocytopenia, resulting in insufficient primary hemostasis.

References

  • 1. Sheats MK, van Wetter AJ, Snyder LA, et al. Disseminated large granular lymphoma in a horse. Equine Vet Educ 2008;20:459463.

  • 2. Mastrorilli C, Cesar F, Joiner K, et al. Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology. Vet Clin Pathol 2015;44:437441.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Herraez P, Berridge B, Marsh P, et al. Small intestine large granular lymphoma in a horse. Vet Pathol 2001;38:223226.

  • 4. Grindem CB, Roberts M, McEntee M, et al. Large granular lymphocyte tumour in a horse. Vet Pathol 1989;26:8688.

  • 5. Quist CF, Harmon B, Mahaffey E, et al. Large granular lymphocyte neoplasia in an aged mare. J Vet Diagn Invest 1994;6:111113.

  • 6. Turinelli V, Marchal T, Ponce F, et al. Aggressive large granular lymphocyte lymphomas in five dogs: a clinical cytohistological and immunological study. Comp Clin Pathol 2005;13:109118.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Roccabianca P, Vernau W, Caniatti M, et al. Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8αα phenotype. Vet Pathol 2006;43:1528.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Frith CH, Ward JM, Chandra M. The morphology, immunohistochemistry, and incidence of hematopoietic neoplasms in mice and rats. Toxicol Pathol 1993;21:206218.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Zambello R, Semenzato G. Large granular lymphocyte disorders: new etiopathogenetic clues as a rationale for innovative therapeutic approaches. Haematologica 2009;94:13411345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Go RS, Tefferi A, Li C, et al. Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia. Blood 2000;96:36443646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Go RS, Lust JA, Phyliky RL. Aplastic anemia and pure red cell aplasia associated with large granular lymphocyte leukemia. Semin Hematol 2003;40:196200.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Address correspondence to Dr. Shrader (sms0059@auburn.edu).
  • View in gallery
    Figure 1—

    Photomicrograph of a cytologic preparation from the right mandibular lymph node of a 14-year-old Missouri Fox Trotter gelding that was evaluated because of chronic weight loss of 1 year's duration and acute epistaxis from the left nostril that occurred 2 days prior. Notice that these cells have scant to moderate amounts of basophilic cytoplasm with few to moderate numbers of large magenta granules, small to intermediate-sized nuclei (approx 1 to 1.5 times the diameter of an RBC), nuclear indentations, condensed chromatin, and absence of visible nucleoli. The background contains numerous cytoplasmic fragments (so-called lymphoglandular bodies). Modifed Wright stain; bar = 15 μm.

  • View in gallery
    Figure 2—

    Photomicrograph of a section of the right mandibular lymph node from the horse in Figure 1. The lymph node is effaced by dense, coalescing, ill-defined nodules of neoplastic lymphocytes. H&E stain; bar = 500μm.

  • View in gallery
    Figure 3—

    Photomicrographs of sections of the right mandibular lymph node from the horse in Figure 1. A—The neoplastic lymphocytes have scant amounts of amphophilic cytoplasm containing hypereosinophilic granules (arrow) and round to irregularly shaped or indented nuclei. H&E stain; bar = 40 μm. B—Following phosphotungstic acid–hematoxylin staining, the intracytoplasmic granules appear dark blue (arrow). Phosphotungstic acid–hematoxylin stain; bar = 30 μm. C—The cytoplasmic immunoreactivity with anti-CD3 antibody (brown labeling) of almost all neoplastic cells identifies them as T lymphocytes. Immunohistochemical CD3-specifc stain, diaminobenzidine chromogen, and hematoxylin counterstain; bar = 30 μm.

  • 1. Sheats MK, van Wetter AJ, Snyder LA, et al. Disseminated large granular lymphoma in a horse. Equine Vet Educ 2008;20:459463.

  • 2. Mastrorilli C, Cesar F, Joiner K, et al. Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology. Vet Clin Pathol 2015;44:437441.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Herraez P, Berridge B, Marsh P, et al. Small intestine large granular lymphoma in a horse. Vet Pathol 2001;38:223226.

  • 4. Grindem CB, Roberts M, McEntee M, et al. Large granular lymphocyte tumour in a horse. Vet Pathol 1989;26:8688.

  • 5. Quist CF, Harmon B, Mahaffey E, et al. Large granular lymphocyte neoplasia in an aged mare. J Vet Diagn Invest 1994;6:111113.

  • 6. Turinelli V, Marchal T, Ponce F, et al. Aggressive large granular lymphocyte lymphomas in five dogs: a clinical cytohistological and immunological study. Comp Clin Pathol 2005;13:109118.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Roccabianca P, Vernau W, Caniatti M, et al. Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8αα phenotype. Vet Pathol 2006;43:1528.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Frith CH, Ward JM, Chandra M. The morphology, immunohistochemistry, and incidence of hematopoietic neoplasms in mice and rats. Toxicol Pathol 1993;21:206218.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Zambello R, Semenzato G. Large granular lymphocyte disorders: new etiopathogenetic clues as a rationale for innovative therapeutic approaches. Haematologica 2009;94:13411345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Go RS, Tefferi A, Li C, et al. Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia. Blood 2000;96:36443646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Go RS, Lust JA, Phyliky RL. Aplastic anemia and pure red cell aplasia associated with large granular lymphocyte leukemia. Semin Hematol 2003;40:196200.

    • Crossref
    • Search Google Scholar
    • Export Citation

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