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Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
Published on: January 9, 2019
High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas
Stratos Papakonstantinou1, Peter James O'Brien
1Veterinary Pathobiology Section, School of Veterinary Medicine, University College Dublin, Ireland.
Insights
High content imaging (HCI) offers a semi-automated method to diagnose canine lymphoma by analyzing lymphocyte morphology and immunophenotype. This advanced technique accurately distinguishes malignant from benign cells, aiding in prognosis.
Area of Science:
- Veterinary diagnostics
- Cellular imaging and analysis
- Canine oncology
Background:
- Canine lymphoma diagnosis relies on manual assessment of lymphocyte size.
- Immunophenotyping is crucial for prognosis but requires dual-antibody labeling.
- High content imaging (HCI) is an emerging technology for automated microscopy and image analysis.
Purpose of the Study:
- To test if HCI can semi-automate quantitative diagnosis of canine lymphoma.
- To determine if HCI can simultaneously provide immunophenotypic prognosis.
- To identify novel morphometric parameters for lymphoma diagnosis.
Main Methods:
- Lymphocytes from dogs were analyzed using HCI.
- Cells were stained with antibodies for T and B cell identification (CD3, CD21) and Hoechst-33342.
- Key morphological parameters including cell area, nuclear area, cytoplasmic area, and roundness were quantified.
Main Results:
- HCI effectively differentiated malignant from benign lymphocytes.
- Significant differences in cell area, cytoplasmic area, nuclear displacement, and roundness were observed between control and lymphoma groups (P < 0.05).
- HCI provided simultaneous immunophenotypic information.
Conclusions:
- HCI successfully identified novel morphometric parameters for diagnosing canine lymphoma.
- The technology enables simultaneous determination of prognostic immunophenotype.
- HCI presents a promising tool for objective and efficient canine lymphoma diagnostics.
Background:
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.
Objective:
We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis.
Methods:
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.
Results:
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).
Conclusion:
HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.
