Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.
Abstract: Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8 and CD21 were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.
Copyright © 2020 Elsevier B.V. All rights reserved.
Publication Date: 2020-05-13 PubMed ID: 32438246DOI: 10.1016/j.vetimm.2020.110062Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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This research investigates how different methods for enriching leukocytes (white blood cells) in horse blood samples can impact cell characteristics and function. It is found that the technique used can significantly affect cell recovery, viability, and certain other parameters.
Objective of the Research
- The main aim of this study was to compare two different techniques routinely used to enhance the concentration of leukocytes in blood samples from horses. Any variations in results could potentially affect data interpretation in a wide array of immunological tests.
Methods Employed
- The research compared a common blood dilution-leukocyte enrichment technique using a medium known as Histopaque (His) to a modified technique using a separation medium referred to as Syr-LSM.
- The study used peripheral blood from 12 healthy horses for comparison.
- The parameters assessed included cell recovery per milliliter of blood, cell viability, and leukocyte enrichment purity. Further checks were performed on leukocyte cell marker subset phenotype, spontaneous proliferation of leukocytes, mitogen-induced proliferation, and TNFα concentrations.
Key Findings
- The modified Syr-LSM technique resulted in significantly higher leukocyte cell recovery and viability when compared to the common His technique.
- However, the His technique was associated with a significant rise in the percentage of CD8 and CD21 cells and had induced proliferation.
- In spite of these variations, leukocyte cell purity and TNFα concentration were similar for both techniques after further cultivation for 72 hours.
Conclusion and Implications
- The study concludes that the method chosen to enrich leukocytes in a blood sample can significantly bear upon the outcomes of immunological assays.
- This could alter the way data is understood and interpreted, showcasing the need for standardising leukocyte enrichment technique or at least understanding these impacts while interpreting results.
Cite This Article
APA
Connelly C, Norton NA, Hurley DJ, Hart KA, Meichner K, Gogal RM.
(2020).
Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.
Vet Immunol Immunopathol, 225, 110062.
https://doi.org/10.1016/j.vetimm.2020.110062 Publication
Researcher Affiliations
- Department of Veterinary Biosciences and Diagnostic Imaging, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA.
- Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Rm. 1703, College of Veterinary Medicine, 501 D.W. Brooks Drive Athens, GA 30602, USA.
- Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA.
- Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Rm. 1703, College of Veterinary Medicine, 501 D.W. Brooks Drive Athens, GA 30602, USA.
- Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA.
- Department of Veterinary Biosciences and Diagnostic Imaging, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA. Electronic address: rgogal@uga.edu.
MeSH Terms
- Animals
- Blood Cells / cytology
- Blood Cells / drug effects
- Blood Cells / immunology
- Cell Separation / methods
- Cell Separation / veterinary
- Cell Survival
- Female
- Horses
- Leukocytes / cytology
- Leukocytes / drug effects
- Leukocytes / immunology
- Lymphocyte Activation / drug effects
- Male
- Mitogens / pharmacology
- Tumor Necrosis Factor-alpha / analysis
Citations
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