Abstract
Flow cytometry-based immunophenotyping plays a critical role in the diagnosis, classification, staging, and monitoring of hematopoietic malignancies. However, its application in feline medicine remains limited primarily because of the scarcity of species-specific antibodies. Existing studies in cats predominantly focus on analyzing lymphocyte subpopulations in lymph nodes, with fewer investigations conducted on bone marrow and peripheral blood. This study aimed to assess the utility of peripheral blood samples from healthy cats for quantifying lymphocyte subpopulations by flow cytometry to provide basic data useful for future studies on animals with diseases. Using specific antibody panels, we successfully quantified lymphocyte subpopulations in 15 healthy cats. Total leukocytes (CD18+) accounted for a median of 89.7% of peripheral blood cells. Among lymphocytes, CD5+ T cells were the predominant subset, followed by CD21+ B cells. Among T cells, CD4+ helper cells outnumbered CD8+ cytotoxic cells. Notably, CD18 expression exhibited a biphasic pattern: B lymphocytes showed lower fluorescence intensity compared with T lymphocytes. In some cases, three distinct fluorescence peaks suggested further heterogeneity within the T-cell population. To the best of our knowledge, this is the first study to clearly identify a biphasic expression pattern of CD18 in the peripheral blood lymphocytes of cats. These findings reveal the complexity of immune cell marker expression in the peripheral blood of felines and support the broader application of flow cytometry in feline immunological and diagnostic research. The establishment of these baseline immunophenotypic profiles in healthy cats is crucial for improving the diagnosis and monitoring of feline hematopoietic diseases, ultimately contributing to better clinical management and therapeutic strategies.
References
Araghi, A., Nassiri, S. M., Atyabi, N., Rahbarghazi, R., & Mohammadi, E. (2014). Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies. Journal of Feline Medicine and Surgery, 16(4), 265-274. https://doi.org/10.1177/1098612X13505575. PMid:24065708.
Bęczkowski, P. M., Litster, A., Lin, T. L., Mellor, D. J., Willett, B. J., & Hosie, M. J. (2015). Contrasting clinical outcomes in two cohorts of cats naturally infected with feline immunodeficiency virus (FIV). Veterinary Microbiology, 176(1-2), 50-60. https://doi.org/10.1016/j.vetmic.2014.12.023. PMid:25595267.
Béné, M. C., Le Bris, Y., Robillard, N., Wuillème, S., Fouassier, M., & Eveillard, M. (2016). Flow cytometry in hematological nonmalignant disorders. International Journal of Laboratory Hematology, 38(1), 5-16. https://doi.org/10.1111/ijlh.12438. PMid:26542092.
Byrne, K. M., Kim, H. W., Chew, B. P., Reinhart, G. A., & Hayek, M. G. (2000). A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes. Veterinary Immunology and Immunopathology, 73(2), 167-182. https://doi.org/10.1016/S0165-2427(99)00163-4. PMid:10690932.
Campbell, D. J., Rawlings, J. M., Koelsch, S., Wallace, J., Strain, J. J., & Hannigan, B. M. (2004). Age-related differences in parameters of feline immune status. Veterinary Immunology and Immunopathology, 100(1-2), 73-80. https://doi.org/10.1016/j.vetimm.2004.03.002. PMid:15182997.
Comazzi, S., Gelain, M. E., Spagnolo, V., Riondato, F., Guglielmino, R., & Sartorelli, P. (2006). Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45. Veterinary Clinical Pathology, 35(1), 47-54. https://doi.org/10.1111/j.1939-165X.2006.tb00088.x. PMid:16511791.
Evans, S. J. M. (2023). Flow cytometry in veterinary practice. The Veterinary Clinics of North America. Small Animal Practice, 53(1), 89-100. https://doi.org/10.1016/j.cvsm.2022.07.008. PMid:36270838.
Guzera, M., Cian, F., Leo, C., Winnicka, A., & Archer, J. (2016). The use of flow cytometry for immunophenotyping lymphoproliferative disorders in cats: A retrospective study of 19 cases. Veterinary and Comparative Oncology, 14(Suppl 1), 40-51. https://doi.org/10.1111/vco.12098. PMid:24890438.
Hofmann-Lehmann, R., Holznagel, E., Ossent, P., & Lutz, H. (1997). Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: Hematology, clinical chemistry, and lymphocyte subsets. Clinical and Diagnostic Laboratory Immunology, 4(1), 33-42. https://doi.org/10.1128/cdli.4.1.33-42.1997. PMid:9008278.
Jaroensong, T., Piamwaree, J., & Sattasathuchana, P. (2022). Effects of chemotherapy on hematological parameters and CD4+/CD8+ ratio in cats with mediastinal lymphoma and seropositive to feline leukemia virus. Animals, 12(3), 223. https://doi.org/10.3390/ani12030223. PMid:35158547.
Knotek, Z., Toman, M., & Faldyna, M. (2000). Clinical and immunological characteristics of cats affected by feline infectious peritonitis. Acta Veterinaria Brno, 69(1), 51. https://doi.org/10.2754/avb200069010051.
Litster, A., Lin, J. M., Nichols, J., & Weng, H. Y. (2014). Diagnostic utility of CD4%:CD8 low% T-lymphocyte ratio to differentiate feline immunodeficiency virus (FIV)-infected from FIV-vaccinated cats. Veterinary Microbiology, 170(3-4), 197-205. https://doi.org/10.1016/j.vetmic.2014.01.014. PMid:24559863.
Manohar, S. M., Shah, P., & Nair, A. (2021). Flow cytometry: Principles, applications and recent advances. Bioanalysis, 13(3), 181-198. https://doi.org/10.4155/bio-2020-0267. PMid:33543666.
Martini, V., Bernardi, S., Marelli, P., Cozzi, M., & Comazzi, S. (2018). Flow cytometry for feline lymphoma: A retrospective study regarding pre-analytical factors possibly affecting the quality of samples. Journal of Feline Medicine and Surgery, 20(6), 494-501. https://doi.org/10.1177/1098612X17717175. PMid:28675320.
Martini, V., Bernardi, S., Giordano, A., & Comazzi, S. (2020). Flow cytometry expression pattern of CD44 and CD18 markers on feline leukocytes. Journal of Veterinary Diagnostic Investigation, 32(5), 706-709. https://doi.org/10.1177/1040638720945670. PMid:32718218.
McKinnon, K. M. (2018). Flow cytometry: An overview. Journal of Current Protocols in Immunology, 120, 5.1.1-5.1.11. https://doi.org/10.1002/cpim.40.
Rout, E. D., Labadie, J. D., Curran, K. M., Yoshimoto, J. A., Avery, A. C., & Avery, P. R. (2020). Immunophenotypic characterization and clinical outcome in cats with lymphocytosis. Journal of Veterinary Internal Medicine, 34(1), 105-116. https://doi.org/10.1111/jvim.15650. PMid:31693230.
Rütgen, B. C., Baszler, E., Weingand, N., Wolfesberger, B., Baumgartner, D., Hammer, S. E., Groiss, S., FuchsBaumgartinger, A., Saalmuller, A., & Schwendenwein, I. (2022). Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats. Journal of Feline Medicine and Surgery, 24(2), 77-90. https://doi.org/10.1177/1098612X211005310. PMid:33908810.
Sánchez-Solé, R., Pedreira, G., Venzal, J. M., Fonseca-Alves, C. E., & Serdio, P. P. (2022). The use of flow cytometry for diagnosis and immunophenotyping in chronic lymphocytic leukemia in a dog: Clinical case report. Open Veterinary Journal, 12(6), 868-876. https://doi.org/10.5455/OVJ.2022.v12.i6.13. PMid:36650866.
Sánchez-Solé, R., Mosquillo, F., Jark, P., Breijo, M., & Pessina, P. (2021). Hematological and biochemical profiles of canine CD45- T lymphomas are different from other immunophenotypes. Open Veterinary Journal, 11(4), 734-746. https://doi.org/10.5455/OVJ.2021.v11.i4.26. PMid:35070871.
Shirani, D., Nassiri, S. M., Aldavood, S. J., Seddigh, H. S., & Fathi, E. (2011). Acute erythroid leukemia with multilineage dysplasia in a cat. The Canadian Veterinary Journal = La Revue Vétérinaire Canadienne, 52(4), 389-363. PMid:21731091.
Vapniarsky, N., Simpson, D. L., Arzi, B., Taechangam, N., Walker, N. J., Garrity, C., Bulkeley, E., & Borjesson, D. L. (2020). Histological, immunological, and genetic analysis of feline chronic gingivostomatitis. Frontiers in Veterinary Science, 7, 310. https://doi.org/10.3389/fvets.2020.00310. PMid:32582783.
Willett, B. J., Hosie, M. J., Callanan, J. J., Neil, J. C., & Jarrett, O. (1993). Infection with feline immunodeficiency virus is followed by the rapid expansion of a CD8+ lymphocyte subset. Immunology, 78(1), 1-6. PMid:8094707.

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Copyright (c) 2025 Patricia Lindiman, Rosina Sánchez Solé, Paula Pessina, María Florencia Mosquillo, María Florencia Mosquillo

