Effect of Anticoagulants on Blood Cell Counts, Cell Morphology, and Leukocyte Immune Functions of Rainbow Trout (Oncorhynchus mykiss)

Hematological and immunological parameters may be influenced by various factors, including anticoagulants used to collect blood samples. This study aimed to evaluate the effect of three different anticoagulants (Li-Heparin, K₃EDTA, and ACD-A) on rainbow trout blood cell count and morphology, leukocyte viability, ROS production, and phagocytosis. These evaluations were assessed through a combined approach based on flow cytometry and microscopy. Blood cell counts revealed differences in erythrocyte and thrombocyte count between anticoagulants (<i>p</i> < 0.001 and <i>p</i> < 0.01, respectively). Whole-blood samples collected in Li-Heparin displayed no hemolysis, in contrast to ACD-A and K₃EDTA. A lower percentage of live cells and a higher percentage of apoptotic and dead cells were observed in K₃EDTA compared to ACD-A (<i>p</i> < 0.05). Significantly lower ROS levels were observed in myeloid cells in Li-Heparin and ACD-A, compared to K₃EDTA samples (<i>p</i> < 0.05). Overall, Li-Heparin better preserves rainbow trout blood cells in terms of hematological count, cell morphology, and ROS production and does not negatively affect the phagocytosis ability of leukocytes. Furthermore, the combined use of both flow cytometry and microscopy can provide a deeper insight into morpho-functional characteristics of leukocytes. Finally, we propose a flow cytometric assay to evaluate rainbow trout leukocyte viability, ROS production, and phagocytosis.