Vallejos-Vidal Eva, Santillán-Araneda María J, Goldstein Merari, Solarte-Murillo Laura V, Maisey Kevin, Reyes-Cerpa Sebastián, Vidal Mabel, Reyes-Lopez Felipe E
Fish Health and Integrative Physiogenomics Research Team, Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Avenida Libertador Bernardo O'Higgins 3363, Edificio de Investigación Eduardo Morales, Estación Central, Santiago, Chile; Núcleo de Investigaciones Aplicadas en Ciencias Veterinarias y Agronómicas, Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Avenida Walker Martínez 1360, La Florida, 8242125, Santiago de, Chile.
Fish Health and Integrative Physiogenomics Research Team, Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Avenida Libertador Bernardo O'Higgins 3363, Edificio de Investigación Eduardo Morales, Estación Central, Santiago, Chile.
Fish Shellfish Immunol. 2025 Jun;161:110291. doi: 10.1016/j.fsi.2025.110291. Epub 2025 Mar 20.
Peripheral blood leukocytes (PBL) are critical for understanding systemic immune responses and assessing the organism's health. In immunological studies, particularly with aquaculture-relevant species like Rainbow trout (Oncorhynchus mykiss), ensuring PBL viability during sample storage is important, especially when samples must be transported over long distances. Anticoagulants are essential for preventing blood clotting and preserving cellular integrity; however, their effects on leukocyte populations in salmonids remain poorly studied. This study aimed to evaluate the impact of four anticoagulants-ethylenediaminetetraacetic acid (EDTA), sodium citrate, sodium heparin, and lithium heparin-on the viability of PBL populations in rainbow trout, with a comparative analysis of human PBL. Blood samples were stored for up to seven days, and cell viability and leukocyte population percentages were assessed by flow cytometry at 0, 1-, 2-, 4-, and 7-days post-collection (dpc). Results showed that sodium heparin and lithium heparin were the most effective anticoagulants for preserving trout leukocyte viability, maintaining percentages greater than 70 % up to 4 dpc. In contrast, EDTA and sodium citrate were less effective in maintaining cell viability. In human PBL samples, EDTA was the most effective anticoagulant, with lymphocyte viability exceeding 80 % at 7 dpc. Sodium heparin and lithium heparin also preserved human PBL viability comparably up to 4 dpc, but their efficacy decreased significantly by 7 dpc. These findings highlight the species-specific effects of anticoagulants, recommending heparin-based anticoagulants for long-term leukocyte preservation in rainbow trout (up to 4 dpc). In addition, this study provides valuable information for blood handling protocols in immunological research.
外周血白细胞（PBL）对于理解全身免疫反应和评估机体健康至关重要。在免疫学研究中，尤其是对于与水产养殖相关的物种，如虹鳟（Oncorhynchus mykiss），在样本储存期间确保PBL的活力很重要，特别是当样本必须长途运输时。抗凝剂对于防止血液凝固和保持细胞完整性至关重要；然而，它们对鲑科鱼类白细胞群体的影响仍研究不足。本研究旨在评估四种抗凝剂——乙二胺四乙酸（EDTA）、柠檬酸钠、肝素钠和肝素锂——对虹鳟PBL群体活力的影响，并对人类PBL进行比较分析。血液样本储存长达七天，在采集后0、1、2、4和7天（dpc）通过流式细胞术评估细胞活力和白细胞群体百分比。结果表明，肝素钠和肝素锂是保存鳟鱼白细胞活力最有效的抗凝剂，在4 dpc时保持百分比大于70%。相比之下，EDTA和柠檬酸钠在维持细胞活力方面效果较差。在人类PBL样本中，EDTA是最有效的抗凝剂，在7 dpc时淋巴细胞活力超过80%。肝素钠和肝素锂在4 dpc之前也能同等程度地保存人类PBL活力，但到7 dpc时其效果显著下降。这些发现突出了抗凝剂的物种特异性效应，推荐基于肝素的抗凝剂用于虹鳟白细胞的长期保存（长达4 dpc）。此外，本研究为免疫学研究中的血液处理方案提供了有价值的信息。
