Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats

Abstract

Objectives Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. Methods Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. Results Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. Conclusions and relevance This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.

Flowcytometric data of intermediate-large cell gastrointestinal lymphoma presenting a gross mass in 32 cats – “let them glow in the flow”

“…In dogs and in cats, non-specific binding of neoplastic T cells has been observed. Thus, CD79αcy must be considered a less reliable antibody for labelling B cells than CD21 ( 19 ). The single case of DLBCL exhibiting CD3 positivity in our case series was the only one showing discrepant results with histopathology and clonality testing being monoclonal for IGH-VDJ.…”

“…As there is no definite proved cut off published for a “monomorphic” expression pattern and a reactive pattern in FCM – in our lab we have the cut off with >90% of the gated cells showing the same expression pattern (eg all CD3 + CD21 − CD4 − CD8 − CD18 + CD5 − CD79 − ) – as done in a former publication ( 12 ). The only reference available based on feline normal and mildly reactive peripheral lymph nodes describes a range of: CD3 + 54.81% ± 11.10%, CD5 + 57.39% ± 12.66%, CD21 + 40.42% ± 12.40%, CD79αcy + 30.41% ± 13.49%; 30.88% ± 13.48% CD4 + , 12.91% ± 6.68% CD8 + cells ( 19 ). Cases eventually presenting with <90% uniformity and subpopulations within the reported ranges were not considered as lymphomas.…”

La citometría de flujo, un universo de posibilidades en el ámbito veterinario. Revisión

“…Hay que recordar que existen moléculas que se expresan en varias estirpes celulares, mientras que la expresión de otras es exclusiva de ciertas poblaciones o subpoblaciones celulares, lo que implica que debe hacerse una correcta selección de anticuerpos. Debe tenerse en cuenta que las moléculas que identifican a cada población celular pueden depender de la especie animal con la que se esté trabajando (38)(39)(40)(41)(42) .…”

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“…Though a feline CD3 monoclonal antibody recognizing a surface epitope has been described (26) this antibody is not commercially available and has so far rarely been used due to difficulties with conjugation. The anti-human CD3 monoclonal antibody clone CD3-12, targeting an intracytoplasmic epitope of CD3e and cross-reactive with CD3 molecules of multiple species, has been used and reported with success for the detection of feline T cells, and is commercially available conjugated to several different fluorochromes (27). However, this antibody requires permeabilization for intracytoplasmic staining, a step which we did not want to include in our initial protocol.…”