The Use of Cytochemistry, Immunophenotyping, Flow Cytometry, and In Vitro Differentiation to Determine the Ontogeny of a Canine Monoblastic Leukemia

Abstract

We evaluated the utility of cytochemistry, immunophenotyping, flow cytometry, and in vitro culture with forced differentiation of leukemic cells as diagnostic aids to identify the malignant cell ontogeny in a dog with leukemia. A tentative diagnosis of monoblastic leukemia was established by microscopic examination of Romanowsky-stained blood smears and bone marrow aspirate smears. This diagnosis also was supported by the light scatter signature that identified the blast cells as large, non-granular monocytic cells using a CellDyn 3500 automated hematology analyzer; as well as by the detection of N-butyrate esterase and the lack of choloroacetate esterase or leukocyte peroxidase by cytochemical staining. Subsequently, leukemic cells were isolated from the dog's peripheral blood and placed into tissue culture or cryopreserved. The leukemic cells grew in suspension cultures and proliferated spontaneously for up to 4 days. By day 7, proliferation was negligible. Upon culture with conditioned supernatant using mitogen-stimulated human T cells as a source of cytokines, an increased proportion of cells entered S phase by day 2 of culture; however, proliferation declined markedly by day 4, at which time the cells had apparently differentiated to adherent, vacuolated macrophages. The cytokine-stimulated leukemic cells were positive for the monocyte/macrophage specific markers alpha-1-antitrypsin, alpha-1-antichymotrypsin, lysozyme, CD14, MHC class II, and calprotectin, an antigen found in differentiated macrophages and granulocytes. Despite the strong tendency of the leukemic cells towards monocytic differentiation, our results suggested that they retained some features of a myelomonocytic precursor. These data show that cytochemistry, immunophenotyping, flow cytometry, and in vitro differentiation of canine leukemia cells are useful tools for confirming the lineage of malignant hematopoietic cells.

Sequential Low‐Dose Rate Half‐Body Irradiation and Chemotherapy for the Treatment of Canine Multicentric Lymphoma

“…Staging was done according the World Health Organization criteria 14 . Where possible, staging diagnostics included a CBC, serum biochemistry, urinalysis, thoracic radiographs, abdominal ultrasound examination, BM aspirate evaluation, and tumor immunophenotype (CD3, a Pan T cell and CD79a, b Pan B cell) as described previously 15 …”

A toxicity study of low‐dose rate half‐body irradiation and chemotherapy in dogs with lymphoma

“…All dogs were clinically staged (3–5) at diagnosis by using the World Health Organization criteria for canine lymphoma 12 . Staging included a complete blood count (CBC), chemistry/electrolyte panel, urinalysis, thoracic radiographs, abdominal ultrasound, BM aspirate evaluation and tumour immunophenotype [CD3 (Serotec, Raleigh, NC, USA), Pan T‐cell and CD79a (Dako, Carpinteria, CA, USA), Pan B‐cell] as previously described 13 . Dogs were excluded if previously treated for lymphoma or if they had any concomitant life‐threatening disease.…”

Diagnosis of Canine Lymphoid Neoplasia Using Clonal Rearrangements of Antigen Receptor Genes

“…Samples from University of California at Davis were analyzed by flow cytometry as described in Moore et al 12 Samples from Texas A&M University were phenotyped according to Modiano et al 11 Leukemia samples from CSU were typed as follows: 200 l of whole blood was lysed in 1 ml of lysis buffer (0.15 M NH 4 Cl, 1 M KHCO 3 , 0.1 mM Na 2 EDTA, 1 N HCl to a pH of 7.2-7.4) for 5 minutes at room temperature. The cells were spun, lysed a second time, then resuspended in 200 l of phosphate buffered saline (PBS)-2% FBS.…”