A rapid multiple immunofluorescence method for the simultaneous detection of CD20 and CD3 in canine and feline cytological samples

Abstract

CD20 and CD3 are considered reliable markers for B and T cells, respectively. This study aimed to develop a rapid multiple immunofluorescence (RMIF) method for the detection of CD20 and CD3 on a single cytology slide. Air-dried smears were prepared using samples collected from dogs (n=26) and cats (n=6). Immunosignal detection using the newly developed method required 60 min. Clear immunosignals for CD20 and CD3 were detected in 24 of 26 samples in dogs and in all 6 cats. As the RMIF (CD20/CD3) method can detect markers of both B and T cells simultaneously on a single cytology smear, it would be an efficient tool for the immunophenotyping of canine and feline lymphoma samples.

Keywords: CD20; CD3; immunocytochemistry; immunophenotyping; lymphoma.

Fig. 1.

Lymph node samples stained using the rapid multiple immunofluorescence (RMIF) CD20/CD3 method. A: B-cell lymphoma in a dog. B: T-cell lymphoma in a dog. C: Reactive lymphoid hyperplasia in a dog. Green signal; CD20-Alexa 488, red signal; CD3-Alexa 594, blue signal; DAPI. Samples were prepared using fine needle biopsy. Scale bars=20 µm.

Fig. 2.

Comparison of the rapid multiple immunofluorescence (RMIF) CD20/CD3 and RMIF CD79α/CD3 methods. A & B: Detection of CD20. C & D: Detection of CD79α. A & C: Fine needle biopsy samples from the lymph node of a dog with T-cell lymphoma. B & D: Sediment smear from the chylous effusion of a cat. In the RMIF CD20/CD3 samples, green signals for CD20 were observed in the margins of certain lymphocytes, and negative reactions were clearly observable in most lymphocytes. In the RIMF CD79α/CD3 samples, the weak, nonspecific green signals were detected in multiple lymphocytes. In all panels, a fluorescence filter adapted to Alexa 488 was used. Scale bars=10 µm.