{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T22:57:57Z","timestamp":1791586677559,"version":"4.3.4"},"reference-count":44,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,9,2]],"date-time":"2025-09-02T00:00:00Z","timestamp":1756771200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Agriculture, Food Sovereignty and Forests, project INNOFISHFARM","award":["J89J21004200001"],"award-info":[{"award-number":["J89J21004200001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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 (p &lt; 0.001 and p &lt; 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 (p &lt; 0.05). Significantly lower ROS levels were observed in myeloid cells in Li-Heparin and ACD-A, compared to K3EDTA samples (p &lt; 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.<\/jats:p>","DOI":"10.3390\/cells14171372","type":"journal-article","created":{"date-parts":[[2025,9,2]],"date-time":"2025-09-02T15:02:57Z","timestamp":1756825377000},"page":"1372","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Effect of Anticoagulants on Blood Cell Counts, Cell Morphology, and Leukocyte Immune Functions of Rainbow Trout (Oncorhynchus mykiss)"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0089-5946","authenticated-orcid":false,"given":"Teresina","family":"De Iorio","sequence":"first","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2626-7059","authenticated-orcid":false,"given":"Maria Carmela","family":"Scat\u00e0","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Grandoni","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2963-3365","authenticated-orcid":false,"given":"Giovanna","family":"De Matteis","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5283-7521","authenticated-orcid":false,"given":"Arianna","family":"Martini","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6669-7355","authenticated-orcid":false,"given":"Nicol\u00f2","family":"Tonachella","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Domitilla","family":"Pulcini","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4752-6881","authenticated-orcid":false,"given":"Fabrizio","family":"Capoccioni","sequence":"additional","affiliation":[{"name":"Consiglio per la Ricerca in Agricoltura e l\u2019Analisi Dell\u2019Economia Agraria (CREA), Research Centre for Animal Production and Aquaculture, 00015 Monterotondo, RM, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Seibel, H., Ba\u00dfmann, B., and Rebl, A. (2021). Blood Will Tell: What Hematological Analyses Can Reveal About Fish Welfare. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.616955"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/j.aquaculture.2018.10.030","article-title":"Fish Hematology Analysis as an Important Tool of Aquaculture: A Review","volume":"500","author":"Fazio","year":"2019","journal-title":"Aquaculture"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"737498","DOI":"10.1016\/j.aquaculture.2021.737498","article-title":"Hematological Methods in Fish\u2014Not Only for Beginners","volume":"547","author":"Witeska","year":"2022","journal-title":"Aquaculture"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2942","DOI":"10.1016\/j.sjbs.2022.01.019","article-title":"Hematological and Serum Biochemical Reference Intervals of Rainbow Trout, Oncorhynchus mykiss Cultured in Himalayan Aquaculture: Morphology, Morphometrics and Quantification of Peripheral Blood Cells","volume":"29","author":"Nabi","year":"2022","journal-title":"Saudi J. Biol. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.2331\/fishsci.68.sup2_1135","article-title":"Relationships between Immune and Reproductive Endocrine Systems in Fish","volume":"68","author":"Saha","year":"2002","journal-title":"Fish. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"105","DOI":"10.2754\/avb200473010105","article-title":"Red Blood Cell Indices for Rainbow Trout (Oncorhynchus mykiss Walbaum) Reared in Cage and Raceway Culture","volume":"73","author":"Adamec","year":"2004","journal-title":"Acta Vet. Brno"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1007\/s10695-007-9146-5","article-title":"Effects of Sodium Selenite on Some Biochemical and Hematological Parameters of Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792) Exposed to Pb2+ and Cu2+","volume":"34","author":"Ates","year":"2008","journal-title":"Fish Physiol. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.anifeedsci.2011.12.015","article-title":"Fish Immune System and Its Nutritional Modulation for Preventive Health Care","volume":"173","author":"Kiron","year":"2012","journal-title":"Anim. Feed Sci. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6","DOI":"10.3389\/fimmu.2022.835767","article-title":"Assessing Fish Immunotoxicity by Means of In Vitro Assays: Are We There Yet?","volume":"13","author":"Segner","year":"2022","journal-title":"Front. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.jim.2011.06.027","article-title":"Development and Validation of a Flow Cytometric Method to Evaluate Phagocytosis of pHrodoTM Bioparticles\u00ae by Granulocytes in Multiple Species","volume":"390","author":"Neaga","year":"2013","journal-title":"J. Immunol. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Parv, K., Westerlund, N., Merchant, K., Komijani, M., Lindsay, R.S., and Christoffersson, G. (2021). Phagocytosis and Efferocytosis by Resident Macrophages in the Mouse Pancreas. Front. Endocrinol., 12.","DOI":"10.3389\/fendo.2021.606175"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Scat\u00e0, M.C., Alhussien, M.N., Grandoni, F., Reale, A., Zampieri, M., Hussen, J., and De Matteis, G. (2023). Hyperthermia-Induced Changes in Leukocyte Survival and Phagocytosis: A Comparative Study in Bovine and Buffalo Leukocytes. Front. Vet. Sci., 10.","DOI":"10.3389\/fvets.2023.1327148"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1322","DOI":"10.1111\/j.1095-8649.2011.03118.x","article-title":"The Efficacy of Field Techniques for Obtaining and Storing Blood Samples from Fishes","volume":"79","author":"Clark","year":"2011","journal-title":"J. Fish Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2708","DOI":"10.1002\/eji.202170126","article-title":"Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)","volume":"51","author":"Cossarizza","year":"2021","journal-title":"Eur. J. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.fsi.2019.10.006","article-title":"Advances in Salmonid Fish Immunology: A Review of Methods and Techniques for Lymphoid Tissue and Peripheral Blood Leucocyte Isolation and Application","volume":"95","author":"Lulijwa","year":"2019","journal-title":"Fish Shellfish Immunol."},{"key":"ref_16","first-page":"615","article-title":"Effect of Three Different Anticoagulants and Storage Time on Haematological Parameters of Mugil cephalus (Linneaus, 1758)","volume":"14","author":"Faggio","year":"2014","journal-title":"Turk. J. Fish. Aquat. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"101398","DOI":"10.1016\/j.tice.2020.101398","article-title":"Comparative Evaluation of Two Anticoagulants Used for the Analysis of Haematological, Biochemical Parameters and Blood Cell Morphology of Himalayan Snow Trout, Schizopyge plagiostomus","volume":"67","author":"Sheikh","year":"2020","journal-title":"Tissue Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s10499-018-0308-5","article-title":"The Effect of Dipotassium EDTA and Lithium Heparin on Hematologic Values of Farmed Brown Trout Salmo trutta (L.) Spawners","volume":"27","author":"Kasprzak","year":"2019","journal-title":"Aquac. Int."},{"key":"ref_19","first-page":"239","article-title":"Effects of Two Commonly Used Anticoagulants on Haematology and Erythrocyte Morphology of Rainbow Trout (Oncorhynchus mykiss)","volume":"2","author":"Maqbool","year":"2014","journal-title":"Int. J. Fish. Aquat. Stud."},{"key":"ref_20","first-page":"99","article-title":"Disodium EDTA Used as Anticoagulant Causes Hemolysis in Common Carp Blood","volume":"35","author":"Witeska","year":"2011","journal-title":"Turk. J. Vet. Anim. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1053\/j.tcam.2011.02.003","article-title":"Common Laboratory Artifacts Caused by Inappropriate Sample Collection and Transport: How to Get the Most out of a Sample","volume":"26","author":"Gilor","year":"2011","journal-title":"Top. Companion Anim. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/0300-9629(85)90683-8","article-title":"The Effect of Anticoagulants on Blennius pholis L. Leucocytes","volume":"80","author":"Mainwaring","year":"1985","journal-title":"Comp. Biochem. Physiol.\u2014Part A Physiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.cbpc.2007.04.004","article-title":"The Effects of Anticoagulants on Hematological Indices and Blood Cell Morphology of Common Carp (Cyprinus carpio L.)","volume":"146","author":"Walencik","year":"2007","journal-title":"Comp. Biochem. Physiol.\u2014C Toxicol. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1111\/j.1095-8649.1988.tb05474.x","article-title":"Effects of Sampling Conditions on Selected Blood Variables of Rainbow Trout, Salmo gairdneri Richardson","volume":"33","author":"Korcock","year":"1988","journal-title":"J. Fish Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/0306-4492(77)90074-0","article-title":"Observations on Some Effects of Disodium Ethylenediamine Tetra-Acetate and Heparin on Fish Blood","volume":"57","author":"Smit","year":"1977","journal-title":"Comp. Biochem. Physiol. Part C Comp."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1093\/ajcp\/55.5.565","article-title":"Use of Adenine-ACD Solution in Long Term Storage of Blood","volume":"55","author":"Sussman","year":"1971","journal-title":"Am. J. Clin. Pathol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1007\/s40139-019-00192-8","article-title":"Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications","volume":"7","author":"Betsou","year":"2019","journal-title":"Curr. Pathobiol. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"110291","DOI":"10.1016\/j.fsi.2025.110291","article-title":"Comparison of anticoagulant vacutainer blood collection tubes on rainbow trout (Oncorhynchus mykiss) leukocyte viability during long-term storage","volume":"161","author":"Goldstein","year":"2025","journal-title":"Fish Shellfish Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.dci.2018.07.010","article-title":"Characterisation of Rainbow Trout Peripheral Blood Leucocytes Prepared by Hypotonic Lysis of Erythrocytes, and Analysis of Their Phagocytic Activity, Proliferation and Response to PAMPs and Proinflammatory Cytokines","volume":"88","author":"Hu","year":"2018","journal-title":"Dev. Comp. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Aizawa, H., Kawabata, H., Sato, A., Masuki, H., Watanabe, T., Tsujino, T., Isobe, K., Nakamura, M., Nakata, K., and Kawase, T. (2020). A Comparative Study of the Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency. Biomedicines, 8.","DOI":"10.3390\/biomedicines8030042"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1080\/09537100802364076","article-title":"The Effect of Ex Vivo Anticoagulants on Whole Blood Platelet Aggregation","volume":"20","author":"Kalb","year":"2009","journal-title":"Platelets"},{"key":"ref_32","first-page":"1","article-title":"Ex Vivo Anticoagulants Affect Human Blood Platelet Biomechanics with Implications for High-Throughput Functional Mechanophenotyping","volume":"5","author":"Sachs","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1002\/cyto.990130111","article-title":"Flow Cytometric Analysis of Whole Blood Lysis, Three Anticoagulants, and Five Cell Preparations","volume":"13","author":"Carter","year":"1992","journal-title":"Cytometry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"547","DOI":"10.17221\/98\/2019-VETMED","article-title":"Optimisation of the Lymphocyte Proliferation Assay in Rainbow Trout (Oncorhynchus mykiss)","volume":"64","author":"Minarova","year":"2019","journal-title":"Vet. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.mrgentox.2008.11.010","article-title":"Establishment of Experimental Conditions for Preserving Samples of Fish Blood for Analysis with Both Comet Assay and Flow Cytometry","volume":"673","author":"Ramsdorf","year":"2009","journal-title":"Mutat. Res.\u2014Genet. Toxicol. Environ. Mutagen."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.pharmthera.2015.11.006","article-title":"Pharmacological Models and Approaches for Pathophysiological Conditions Associated with Hypoxia and Oxidative Stress","volume":"158","author":"Herrera","year":"2016","journal-title":"Pharmacol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1667\/RR3299","article-title":"Standardization of a Fluorometric Assay for Measuring Oxidative Stress in Irradiated Cells","volume":"163","author":"Wan","year":"2005","journal-title":"Radiat. Res."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Wang, J., Lei, P., Gamil, A.A.A., Lagos, L., Yue, Y., Schirmer, K., Mydland, L.T., \u00d8verland, M., Krogdahl, \u00c5., and Kortner, T.M. (2019). Rainbow Trout (Oncorhynchus mykiss) Intestinal Epithelial Cells as a Model for Studying Gut Immune Function and Effects of Functional Feed Ingredients. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.00152"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Park, Y., Abihssira-Garc\u00eda, I.S., Thalmann, S., Wiegertjes, G.F., Barreda, D.R., Olsvik, P.A., and Kiron, V. (2020). Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00203"},{"key":"ref_40","first-page":"1192","article-title":"Anticoagulant Induced Leukoagglutination","volume":"29","year":"2008","journal-title":"Saudi Med. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1093\/ajcp\/103.3.338","article-title":"Ethylenediamine Tetraacetic Acid-Associated Leukoagglutination","volume":"103","author":"Deol","year":"1995","journal-title":"Am. J. Clin. Pathol."},{"key":"ref_42","first-page":"426","article-title":"Hematology, Transfusion and Cell Therapy Unusual Leukoagglutination: A Rare Haematological Finding","volume":"4","author":"Daves","year":"2020","journal-title":"Hematol. Transfus. Cell Ther."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"112","DOI":"10.4103\/0972-5229.99139","article-title":"Patient Communication in Intensive Care Unit","volume":"16","author":"Haranath","year":"2012","journal-title":"Indian J. Crit. Care Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"298","DOI":"10.17221\/189\/2020-VETMED","article-title":"Optimisation of Phagocytosis Assay in Rainbow Trout (Oncorhynchus mykiss)","volume":"66","author":"Minarova","year":"2021","journal-title":"Vet. Med."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/17\/1372\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:38:16Z","timestamp":1760035096000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/17\/1372"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,2]]},"references-count":44,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["cells14171372"],"URL":"https:\/\/doi.org\/10.3390\/cells14171372","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,2]]}}}