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Home / Equine Research Bank / Vet Clin Pathol / Validation of a flow cytometric assay to detect intraerythro...
Veterinary clinical pathology2021; 50(1); 20-27; doi: 10.1111/vcp.12976

Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses.

Abstract: Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detection methods. Objective: We aimed to validate intraerythrocytic ROS detection by flow cytometry using DCHF-DA in healthy horses. Methods: Erythrocytes from 31 healthy horses were isolated, incubated with DCFH-DA, and either left unstimulated or stimulated with hydrogen peroxide (H O ). For specificity, each cellular component of blood was separated and plotted according to its size and complexity. Samples were run in triplicate for intra-assay precision and five consecutive times for inter-assay repeatability. Stability was determined by analyzing the same sample up to 48 hours after blood collection. The acceptable coefficient of variation (CV) was ≤20%. Results: The intra-assay CV was 1.7% and 13.3%, and the inter-assay CV was 4.8% and 17.8% for unstimulated and stimulated samples, respectively. Unstimulated and stimulated samples were stable for up to 48 and 24 hours, respectively. Stimulated samples had greater fluorescence than unstimulated samples (P < .0001). Conclusions: This flow cytometric assay demonstrated adequate specificity, precision, and stability and is, therefore, a promising technique with multiple applications for studying oxidative stress in horses.
© 2021 American Society for Veterinary Clinical Pathology.
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Publication Date: 2021-03-01 PubMed ID: 33650208DOI: 10.1111/vcp.12976Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article describes the successful validation of a new method for detecting reactive oxygen species (ROS), indicative of oxidative stress, within horse red blood cells using flow cytometry and a special substance called DCFH-DA.

Main Aim

  • The primary aim of this research was to validate the utilization of the DCFH-DA technique for detecting intraerythrocytic reactive oxygen species (ROS) in horses using flow cytometry. This process represents a promising way to study oxidative stress, which can have numerous impacts on equine health.

Oxidative Stress and ROS

  • Oxidative stress refers to the build-up of ROS within the body. These compounds can indirectly cause cell damage and consequently inhibit oxygen delivery.
  • This research was inspired by the need for improved ROS detection methods, as the current methods are usually costly, labor-intensive, and use indirect markers.

The DCFH-DA Technique

  • The researchers selected DCFH-DA as a possible alternative for ROS detection. DCFH-DA turns fluorescent when oxidized by ROS. The resulting fluorescence is proportional to ROS concentrations and is easily detectable through flow cytometry.

Testing Methodology

  • To validate this technique, they isolated erythrocytes (red blood cells) from 31 healthy horses, loaded these cells with DCFH-DA, and subjected the samples to either no stimulation or stimulation with hydrogen peroxide, which promotes ROS formation.
  • To verify the precision of the technique, samples were run in triplicate for intra-assay precision (variability within the same experiment) and five consecutive times for inter-assay repeatability (variability across different experiments).
  • The researchers also assessed stability by analyzing the same sample up to 48 hours after blood collection.

Results and Conclusion

  • The coefficient of variation (CV) for unstimulated samples and stimulated samples was within the acceptable ≤20% range, demonstrating high precision and repeatability of the testing method.
  • Furthermore, the mechanism showed the needed specificity, as stimulated samples demonstrated significantly greater fluorescence compared to unstimulated samples.
  • Based on these findings, the researchers conclude that this flow cytometric assay exhibits sufficient specificity, precision, and stability to be a potentially useful tool in studying oxidative stress in horses.

Cite This Article

APA
Serpa PBS, Woolcock A, Taylor SD, Pires Dos Santos A. (2021). Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses. Vet Clin Pathol, 50(1), 20-27. https://doi.org/10.1111/vcp.12976

Publication

Veterinary clinical pathology
ISSN: 1939-165X
NlmUniqueID: 9880575
Country: United States
Language: English
Volume: 50
Issue: 1
Pages: 20-27

Researcher Affiliations

Serpa, Priscila B S
  • Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.
Woolcock, Andrew
  • Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.
Taylor, Sandra D
  • Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.
Pires Dos Santos, Andrea
  • Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.

MeSH Terms

  • Animals
  • Flow Cytometry / veterinary
  • Fluoresceins
  • Horses
  • Hydrogen Peroxide
  • Oxidative Stress
  • Reactive Oxygen Species

Grant Funding

  • Purdue Veterinary Medicine Equine Research Funding

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Citations

This article has been cited 5 times.
  1. Pasciu V, Nieddu M, Sotgiu FD, Baralla E, Berlinguer F. An Overview on Assay Methods to Quantify ROS and Enzymatic Antioxidants in Erythrocytes and Spermatozoa of Small Domestic Ruminants. Animals (Basel) 2023 Jul 13;13(14).
    doi: 10.3390/ani13142300pubmed: 37508077google scholar: lookup
  2. Taylor SD, Serpa PBS, Santos AP, Hart KA, Vaughn SA, Moore GE, Mukhopadhyay A, Page AE. Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses. J Vet Intern Med 2022 Jul;36(4):1491-1501.
    doi: 10.1111/jvim.16447pubmed: 35698909google scholar: lookup
  3. Shin SW, Kim M, Shin C, Bae H, Park J, Jung DI, Cho KW, Yu D. Understanding the biochemical impact of leukoreduction on canine pRBC storage: a focus on reactive oxygen species and storage lesions. Front Vet Sci 2025;12:1563532.
    doi: 10.3389/fvets.2025.1563532pubmed: 40979360google scholar: lookup
  4. Taylor SD, Hart KA, Vaughn S, Giancola SC, Serpa PBS, Santos AP. Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses. J Vet Intern Med 2024 Jan-Feb;38(1):460-468.
    doi: 10.1111/jvim.16934pubmed: 37948618google scholar: lookup
  5. Zheng J, Liu S, Wang D, Li L, Sarsaiya S, Zhou H, Cai H. Unraveling the functional consequences of a novel germline missense mutation (R38C) in the yeast model of succinate dehydrogenase subunit B: insights into neurodegenerative disorders. Front Mol Neurosci 2023;16:1246842.
    doi: 10.3389/fnmol.2023.1246842pubmed: 37840772google scholar: lookup
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