• 1.

    Fibach E, Dana M. Red blood cells as redox modulators in hemolytic anemia. In: Tombak A, ed. Erythrocyte. London: IntechOpen Ltd, 2019.

  • 2.

    Valko M, Leibfritz D, Moncol J, et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:4484.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Kadiiska MB, Peddada S, Herbert RA, et al. Biomarkers of oxidative stress study VI. Endogenous plasma antioxidants fail as useful biomarkers of endotoxin-induced oxidative stress. Free Radic Biol Med 2015;81:100106.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    McMichael MA. Oxidative stress, antioxidants, and assessment of oxidative stress in dogs and cats. J Am Vet Med Assoc 2007;231:714720.

  • 5.

    de Zwart LL, Meerman JH, Commandeur JN, et al. Biomarkers of free radical damage applications in experimental animals and in humans. Free Radic Biol Med 1999;26:202226.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Chansaisakorn W, Sriphavatsarakorn P, Sopakdittapong P, et al. Oxidative stress and intraerythrocytic concentrations of sodium and potassium in diabetic dogs. Vet Res Commun 2009;33:6775.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Ojaimi C, Kinugawa S, Recchia FA, et al. Oxidant-NO dependent gene regulation in dogs with type I diabetes: impact on cardiac function and metabolism. Cardiovasc Diabetol 2010;9:43.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Kendall A, Woolcock A, Brooks A, et al. Glutathione peroxidase activity, plasma total antioxidant capacity, and urinary F2-isoprostanes as markers of oxidative stress in anemic dogs. J Vet Intern Med 2017;31:17001707.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Pesillo SA, Freeman LM, Rush JE. Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immune-mediated hemolytic anemia. Am J Vet Res 2004;65:16211624.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Kogika MM, Lustoza MD, Hagiwara MK, et al. Evaluation of oxidative stress in the anemia of dogs with chronic kidney disease. Vet Clin Pathol 2015;44:7078.

  • 11.

    Silva AC, de Almeida BF, Soeiro CS, et al. Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease. Can J Vet Res 2013;77:136141.

    • Search Google Scholar
    • Export Citation
  • 12.

    Freeman LM, Rush JE, Milbury PE, et al. Antioxidant status and biomarkers of oxidative stress in dogs with congestive heart failure. J Vet Intern Med 2005;19:537541.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Smith A. Homeostasis of heme in health and disease: current aspects of the structural biology of heme-protein interactions and of gene regulation. DNA Cell Biol 2002;21:245249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Gibson JS, Wadud R, Lu D, et al. Oxidative stress and haemolytic anaemia in dogs and cats: a comparative approach. Integr J Vet Biosci 2019;3:15.

    • Search Google Scholar
    • Export Citation
  • 15.

    Fibach E, Rachmilewitz E. The role of oxidative stress in hemolytic anemia. Curr Mol Med 2008;8:609619.

  • 16.

    Grune T, Sommerburg O, Siems WG. Oxidative stress in anemia. Clin Nephrol 2000;53:S18S22.

  • 17.

    Amer J, Goldfarb A, Fibach E. Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells. Eur J Haematol 2003;70:8490.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Kalyanaraman B, Darley-Usmar V, Davies KJA, et al. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 2012;52:16.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Christov A, Hamdheydari L, Grammas P. Detection of reactive oxygen species by flow cytometry. In: Hensley K, Floyd RA, eds. In: Methods in biological oxidative stress. Totowa, NJ: Humana Press Inc, 2003;175184.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 2010;594:5772.

  • 21.

    Fibach E, Dana M. Oxidative stress in paroxysmal nocturnal hemoglobinuria and other conditions of complement-mediated hemolysis. Free Radic Biol Med 2015;88:6369.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    O'Hara DM, Xu Y, Liang Z, et al. Recommendations for the validation of flow cytometric testing during drug development: II assays. J Immunol Methods 2011;363:120134.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Friedrichs KR, Harr KE, Freeman KP, et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol 2012;41:441453.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Amer J, Zelig O, Fibach E. Oxidative status of red blood cells, neutrophils, and platelets in paroxysmal nocturnal hemoglobinuria. Exp Hematol 2008;36:369377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Webb C, Bedwell C, Guth A, et al. Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes. Vet Immunol Immunopathol 2006;112:129140.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Yi X, Liu M, Luo Q, et al. Toxic effects of dimethyl sulfoxide on red blood cells, platelets, and vascular endothelial cells in vitro. FEBS Open Bio 2017;7:485494.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Daly J, Tiersch TR. Sources of variation in flow cytometric analysis of aquatic species sperm: the effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating. Aquaculture 2012;370–371:179188.

    • Crossref
    • Search Google Scholar
    • Export Citation

Advertisement

Development and validation of a flow cytometric assay for detecting reactive oxygen species in the erythrocytes of healthy dogs

Andrew D. Woolcock DVM1, Priscila B. S. Serpa DVM, MSc, DSc2, Andrea P. Santos DVM, MSc, PhD2, John A. Christian DVM, PhD2, and George E. Moore DVM, PhD3
View More View Less
  • 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 2 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
  • | 3 Department of Veterinary Administration, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

Abstract

OBJECTIVE

To validate the use of a flow cytometric assay that uses 2‘,7‘-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs.

ANIMALS

50 healthy adult dogs.

PROCEDURES

Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA.

RESULTS

Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours.

CONCLUSIONS AND CLINICAL RELEVANCE

Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.

Abstract

OBJECTIVE

To validate the use of a flow cytometric assay that uses 2‘,7‘-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs.

ANIMALS

50 healthy adult dogs.

PROCEDURES

Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA.

RESULTS

Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours.

CONCLUSIONS AND CLINICAL RELEVANCE

Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.

Supplementary Materials

    • Supplementary Table S1 (PDF 117 kb)

Contributor Notes

Drs. Woolcock and Serpa contributed equally to this work.

Address correspondence to Dr. Woolcock (awoolcoc@purdue.edu).