Case # 51 Peripheral blood smear from a dog Sayuri Hayakawa1 Emily C. Graff1, Elizabeth G. Welles1, Cinzia Mastrorilli1, Peter W. Christopherson1, Elizabeth Spangler1, Lauren Reid2 1
Department of Pathobiology, 2 Department of Clinical Sciences Auburn University College of Veterinary Medicine Permission granted only for viewing on SEVPAC website
Signalment and history 8-year-old, M/N, Weimaraner Presented for a possible splenic mass Lethargic and anorexic for 3 days
Physical exam:
- Bright, alert, responsive and energetic - T 102.9F, Pulse 120/minute, Panting, CRT left) - No abnormalities on abdominal palpation Permission granted only for viewing on SEVPAC website
Patient’s values
Reference interval
30.3 ↓
37 – 55
MCV (fL)
70.3
60 – 77
MCHC (g/dL)
33.8
32 – 36
86,100 ↑
0 – 60,000
74,000 ↓ / clumped
164,000 – 510,000
24.1 ↑
8.4 – 13
64,400 ↑
6,000 – 17,000
5,796
3,000 – 11,400
Lymphocytes (/μL)
52,808 ↑
1,000 – 4,000
Monocytes (/μL)
5,796 ↑
150 – 1,350
CBC
Hct (%)
Reticulocytes (/μL) Platelets (/uL) MPV (fL) WBC (/μL) Neutrophils (/μL)
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Cell size
Cell size
Leukocyte scattergram from Advia120
Peroxidase activity
- Lymphocytes - Large unstained cells
Nuclear configuration
- Mononuclear cells - Lysis-resistant cells
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Blood smear
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Blood smear
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•Abdominal ultrasound - Enlarged liver with variable echogenicity - Enlarged spleen with normal echogenicity
•Cytology - Multiple organ involvement of neoplastic LGLs - Bone marrow, liver, spleen and left mandibular lymph node
•Diagnosis Large granular lymphocyte (LGL) leukemia/lymphoma (stage V) Permission granted only for viewing on SEVPAC website
Large granular lymphocytes (LGLs) •Unique morphologic features: – Cytoplasmic azurophilic granules - At least 3 granules, 0.5µm or greater in diameter
– Contain a variety of enzymes and substances – Induce cell death
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Enzymes • Perforin - 534 aa, MW 60,000kDa - Form transmembrane channels - Facilitate delivery of toxic substances (granzymes)
• Granzymes - Serine proteases - Many subgroups; A and B are the major components
Granzyme A - Cause cell death by caspase independent pathway
Granzyme B - Cause apoptosis by various pathways - Directly activate caspase 3 - Mitochondrial pathway through Smac/DIABLO Permission granted only for viewing on SEVPAC website
Cell types •Cytotoxic T cells – αβ T cells and γδ T cell – Express CD3/TCR complex and CD8
•NK cells – Do not express TCR and have germline configuration of T cell receptor genes – No commercially available markers to detect NK cells in dogs
•Morphologically similar •Most canine LGL leukemia originates from spleen – Express leukointegrin αdβ2 (CD11d) – Almost exclusively on the cells in the red pulp
•Bone marrow involvement is only seen in advanced stages Permission granted only for viewing on SEVPAC website
LGL leukemia in people Clinical course varies depending on the cell type •Cytotoxic T cell LGL leukemia - Slow progressive disease - Treatment is generally required only when clinical signs, anemia or neutropenia become evident
•NK cell LGL leukemia - Aggressive disease - Resistant to most chemotherapy protocols - Poor prognosis
LGL leukemia in dogs Clinical course has not been evaluated based on the cell type - Poor prognosis - Respond poorly to chemotherapy Permission granted only for viewing on SEVPAC website
Case Summary • LGL leukemia/lymphoma (stage V) - 3 days clinical history, but the disease may have been progressing over time - Initially responded to chemotherapy but lost to follow-up 6 months after presentation
• If additional phenotypic markers become available, differentiation of cell types may aid in the evaluation of therapies
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Acknowledgements • Drs. Welles, Spangler, Christopherson, Graff and Mastrorilli for supervision • Clin Path Lab technicians
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References • • •
• • • • •
1. Wellman ML (2000) Schalm's veterinary hematology. Eds. Feldman BF, Zinkl JG, Jain NC, 5th edition. 642-647. 2. Seelig DM, Avery PR, Kisseberth WC, Modiano JF (2010) Schalm's veterinary hematology. Eds. Weiss DJ, Wardrop KJ. 6th edition. 528-529. 3. Podack ER, Hengartner H, Lichtenheld MG (1991) A central role of perforin in cytolysis? Annu. Rev. Immunol. 9:129-157. 4. Ewen CL, Kane KP, Bleackley RC (2012) Review. A quarter century of granzymes. Cell Death Differ. 19:28-35. 5. Takahashi T, Otani I, Okuda M, Inoue M, Ito K, Sakai M, Koie H, Yamada Y, Watari T, Sato T, Kanayama K, Tokuriki M (2007) Malignant transformation of T-cell large granular lymphocyte leukemia in a dog. J. Vet. Med. Sci. 69(6):677-681. 6. McDonough SP, Moore PF (2000) Clinical, hematologic, and immunophenotypic characterization of canine large granular lymphocytosis. Vet. Pathol. 37:637-646. 7. Krick EL, Little L, Patel R, Shofer FS, Sorenmo K, Clifford CA, Baez JL (2008) Description of clinical and pathological findings, treatment and outcome of feline large granular lymphocyte lymphoma (1996-2004) Vet. Comp. Oncol. 6(2):102-110. 8. Valli VE (2007) Veterinary comparative hematopathology. 1st edition. 275-365.
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