Double-staining immunocytochemistry on Romanowsky-stained smears for CD20 and CD3ε in canine samples

Romanowsky-stained smears (RSSs) can be used to immunophenotype lymphoid neoplasms by immunocytochemistry (ICC). This technique typically involves incubating the antibodies separately. However, there is no description of a manual double-staining ICC (dsICC) to characterize lymphocytes in canine samples. This study aims to develop, optimize, and validate a dsICC assay for two lymphoid markers on canine RSSs. An initial antibody selection phase using four candidate markers for B-cells (CD20, CD21, PAX-5, CD79a) and two candidate markers for T-cells (CD3ε, CD5) was performed on unstained and RSSs. Further, CD20 and CD3ε were applied to RSSs for protocol development and optimization. Ultimately, the technique was applied to an independent set of 28 cytological preparations of canine lymphoid neoplasms for validation. The dsICC interpretation was compared with the immunophenotype provided by immunohistochemistry and/or flow cytometry, and/or to the genotype provided by PCR for antigen receptor rearrangement. The dsICC correctly immunophenotyped 26/28 samples (92.85%), including 19 B-cell lymphomas (19/20; 95%) and 7 T-cell lymphomas (7/8; 87.5%); the two inconclusive cases (7.14%) were one B-cell and one T-cell neoplasm. In most smears, the neoplastic B and T lymphocytes displayed a moderate to strong immunoreaction against CD20 and CD3ε, respectively. Residual non-neoplastic T and B-cells were also immunolabeled. The dsICC showed almost perfect agreement (κ = 0.84) compared to confirmatory results. This immunodiagnostic assay represents a potential alternative in veterinary oncology for canine lymphoma immunophenotyping; it enables diagnosis and immunophenotyping from a single smear and may be suitable for the development of an automated dsICC.