"High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas"

Background: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High Content Imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. Objective: We tested the hypothesis that HCI could semi-automatise the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Methods: Lymphocytes were obtained from lymph nodes of 6 lymphoma-free and 5 lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T- cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: a) cell area, b) nuclear area, c) nuclear displacement, d) cytoplasmic area, e) nucleus to cell area ratio (N/Cell), f) nucleus to cytoplasm ratio (N/Cyt), g) cell roundness. Results: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences without value overlap between groups. Mean/ median/SD for control versus lymphoma samples were: a) cell area (µm² ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, b) nuclear area (µm² ): 33.4/34.8/3.5 versus 40.2/38.5/5, c) cytoplasm area (µm² ): 22.1/216.2 versus 40/38.4/4.9, d) N/Cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30//0.056, f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (p<0.05). Conclusion: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype. © 2014 Clinical Cytometry Society.