High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas.

Abstract

The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists mainly 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. We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Lymphocytes were obtained from lymph nodes of six lymphoma-free and five 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), and (g) cell roundness. HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences and the means for each of the samples were not overlapping between the two groups. Mean/median/SD for control versus lymphoma samples were: (a) cell area (µm(2) ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, (b) nuclear area (µm(2) ): 33.4/34.8/3.5 versus 40.2/38.5/5, (c) cytoplasm area (µm(2) ): 22.1/21/6.2 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, and (g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (P < 0.05 for all). HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.