De Iorio Teresina, Scatà Maria Carmela, Grandoni Francesco, De Matteis Giovanna, Martini Arianna, Tonachella Nicolò, Pulcini Domitilla, Capoccioni Fabrizio
Consiglio per la Ricerca in Agricoltura e l'Analisi Dell'Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy.
Cells. 2025 Sep 2;14(17):1372. doi: 10.3390/cells14171372.
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, KEDTA, 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 ( < 0.001 and < 0.01, respectively). Whole-blood samples collected in Li-Heparin displayed no hemolysis, in contrast to ACD-A and KEDTA. A lower percentage of live cells and a higher percentage of apoptotic and dead cells were observed in KEDTA compared to ACD-A ( < 0.05). Significantly lower ROS levels were observed in myeloid cells in Li-Heparin and ACD-A, compared to KEDTA samples ( < 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.
血液学和免疫学参数可能受到多种因素的影响，包括用于采集血样的抗凝剂。本研究旨在评估三种不同抗凝剂（锂肝素、KEDTA和ACD-A）对虹鳟血细胞计数和形态、白细胞活力、活性氧（ROS）产生以及吞噬作用的影响。这些评估是通过基于流式细胞术和显微镜检查的联合方法进行的。血细胞计数显示不同抗凝剂之间红细胞和血小板计数存在差异（分别<0.001和<0.01）。与ACD-A和KEDTA相比，用锂肝素采集的全血样本未出现溶血现象。与ACD-A相比，KEDTA中活细胞百分比更低，凋亡和死亡细胞百分比更高（<0.05）。与KEDTA样本相比，锂肝素和ACD-A中髓样细胞的ROS水平显著更低（<0.05）。总体而言，就血细胞计数、细胞形态和ROS产生而言，锂肝素能更好地保存虹鳟血细胞，且不会对白细胞的吞噬能力产生负面影响。此外，流式细胞术和显微镜检查的联合使用能够更深入地洞察白细胞的形态功能特征。最后，我们提出一种流式细胞术检测方法来评估虹鳟白细胞活力、ROS产生和吞噬作用。
