Immunophenotyping

Due to the different prognoses associated with T- and B-cell lymphomas, immunophenotyping of neoplastic cells is routinely performed to subtype lymphoma in the dog (Marconato et al., 2013a).

A large panel of specific antibodies is now available for both dogs and cats (Table 5.1) and may work both in ICC and flow cytometry protocols. Each immunophenotyping technique has some pros and cons. One advantage of ICC is that immunolabeling can be performed on stored smears (months after sampling if smears are adequately fixed and frozen). Another advantage is that relatively low numbers of cells are required to determine if cells are positive for a given antigen (Caniatti et al., 1996). Labeling for more than one antigen can be performed on a single slide using multiple fluorescent secondary antibodies or chromogens, but more commonly, one slide is needed for each antigen detected by ICC. Also, ICC is moderately time-consuming and the number of cells evaluated is low in comparison with flow cytometry, possibly affecting the final diagnosis if a mixed population of cells is present. Flow cytometry has several advantages: (1) analysis of very large numbers of cells, which allows for resolution of poorly represented populations of cells (such as in mixed populations, residual disease, or initial disease); (2) detection of multiple cell markers on a single cell; (3) accurate quantitation of positive cells; (4) high reproducibility and accuracy; and (5) extreme rapidity, with results already available in few hours from sampling. As a disadvantage, flow cytometry requires fresh samples and a minimum of 2 ? 10⁶ cells. The issue of storage may be partly overcome by using specific preservatives or freezing media, but these are poorly standardized in veterinary medicine and a fresh sample is preferable (Comazzi & Gelain, 2011).

Table 5.1 List of the most common antibodies validated for immunophenotyping canine and feline hematopoietic diseases.

Different antibodies and clones show different immunoreactivity in different sample and fixation type

Immunophenotypic marker	Cell type identified	Validated in
CD1	Dendritic cells, histiocytes	Dog
CD3ε (T cell receptor)	T cells	Dog, cat
CD4	T helper cells, T regulatory cells (Tregs), neutrophils (dogs)	Dog, cat
CD5 (scavenger receptor)	T cell	Dog, cat
CD8	Cytotoxic T cells	Dog, cat
CD11b	Myeloid lineage	Dog, cat
CD11d	Neutrophils, macrophages, splenic T lymphocytes, granular lymphocytes	Dog, cat
CD14 (LPS-BP receptor)	Monocytes, macrophages	Dog, cat
CD18 (integrin β2)	All leukocytes (higher in granulocytes and monocytes)	Dog, cat
CD19	B cells	Dog
CD20	B cells	Dog
CD21 (complement receptor)	Mature B cells	Dog, cat
CD22	Mature B cells	Dog
CD25 (IL-2RA)	Activated lymphocytes	Dog
CD3 (CD62L)	Hematopoietic precursor cells	Dog
CD41/61 (GPIIb/IIIa)	Platelets and megakaryocytes	Dog
CD44	All leukocytes, erythrocyte precursors	Dog
CD45 (common leukocyte antigen)	All leukocytes	Dog
CD79a (Igα of the B-cell receptor)	B cells	Dog, cat
CD117 (c-Kit)	Mastocytes, some hematopoietic precursor cells	Dog
CD204 (scavenger receptor)	Myeloid cells, histiocytes	Dog
FoxP3	Activated lymphocytes, Tregs	Dog
Ionized calcium-binding adapter molecule 1 (IBA1)	Macrophages	Dog
IgE	Mastocytes, B cells	Dog
MHC class II	Monocytes, macrophages, B cells, some T cell subsets	Dog
Pax5	B cells	Dog, cat
Surface immunoglobulin (IgG or IgM)	Mature B cells	Dog
TCRαβ	Most T cells	Dog
TCRγδ	Splenic or intestinal T cells	Dog

PCR to detect antigen receptor rearrangements (PARR) and specific PCR techniques for diagnosis of infectious diseases may help to solve differentials among neoplastic and reactive diseases (Lana et al., 2006a). Recent papers identified 100% sensitivity and specificity of PARR for known cases of lymphoma vs.

non-lymphoma diagnosed using fine needle aspirates from dogs (Ehrhart et al., 2018). For feline lymphoma some authors reported 78% sensitivity and 81% accuracy for B-cell lymphoma (Welter et al., 2021) but undetermined specificity for T-cell lymphoma (Moore et al., 2005). Therefore, the lineage of neoplastic lymphocytes is assessed by PARR with a sufficient reliability, even though immunophenotyping techniques are the method of choice and provide a higher accuracy rate (Thaleim et al., 2013). If necessary, DNA for PCR techniques may be extracted from stained cytologic smears or from paraffin embedded biopsies. Unfortunately, PCR is time-consuming and expensive and results should be interpreted with caution since false-positive and false-negative results are reported in many different conditions.

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Source: Barger A.M., MacNeill A.L. (Eds.). Small Animal Cytologic Diagnosis: Canine and Feline Disease. CRC Press,2024. — 536 p.. 2024

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