[en] Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two secondary antibodies and two linking-fluorochromes, respectively.
Research Center/Unit :
CORD - Centre de l'Oxygène, Recherche et Développement - ULiège
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Wauters, Jella; Faculty of Veterinary Medicine, Ghent University > Pharmacology, Toxicology and Biochemistry
Franck, Thierry ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Anesthésiologie gén. et pathologie chirurg. des grds animaux
Pille, Frederik
Martens, Ann
Demeyere, Kristel
Sys, Stanislas
Serteyn, Didier ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Anesthésiologie gén. et pathologie chirurg. des grds animaux
Gasthuys, Frank
Meyer, Evelyne; Faculty of Veterinary Medicine, Ghent University > Pharmacology, Toxicology and Biochemistry
Language :
English
Title :
Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research.
Publication date :
2011
Journal title :
Veterinary Immunology and Immunopathology
ISSN :
0165-2427
eISSN :
1873-2534
Publisher :
Elsevier, Amsterdam, Netherlands
Volume :
144
Issue :
3-4
Pages :
417-22
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
IWT - Agency for Innovation by Science and Technology
Commentary :
Copyright (c) 2011 Elsevier B.V. All rights reserved.
Art T., Franck T., Lekeux P., de Moffarts B., Couetil L., Becker M., Kohnen S., Deby-Dupont G., Serteyn D. Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction. Can. J. Vet. Res. 2006, 70:291-296.
Borregaard N., Cowland J.B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997, 89:3503-3521.
Borregaard N., Sorensen O.E., Theilgaard-Monch K. Neutrophil granules: a library of innate immunity proteins. Trends Immunol. 2007, 28:340-345.
Bradley P.P., Priebat D.A., Christensen R.D., Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 1982, 78:206-209.
Brennan M.L., Anderson M.M., Shih D.M., Qu X.D., Wang X., Mehta A.C., Lim L.L., Shi W., Hazen S.L., Jacob J.S., Crowley J.R., Heinecke J.W., Lusis A.J. Increased atherosclerosis in myeloperoxidase-deficient mice. J. Clin. Invest. 2001, 107:419-430.
Edwards S.W., Hughes V., Barlow J., Bucknall R. Immunological detection of myeloperoxidase in synovial fluid from patients with rheumatoid arthritis. Biochem. J. 1988, 250:81-85.
Faldyna M., Zelnickova P., Stepanova H., Ondracek J., Kovaru F. Intracellular cytokine detection by flow cytornetry in pigs: fixation, permeabilization and cell surface staining. J. Immunol. Methods 2007, 327:18-29.
Fietz S., Bondzio A., Moschos A., Hertsch B., Einspanier R. Measurement of equine myeloperoxidase (MPO) activity in synovial fluid by a modified MPO assay and evaluation of joint diseases - an initial case study. Res. Vet. Sci. 2008, 84:347-353.
Flemmig J., Lessig J., Reibetanz U., Dautel P., Arnhold J. Non-vital polymorphonuclear leukocytes express myeloperoxidase on their surface. Cell. Physiol. Biochem. 2008, 21:287-296.
Frampton M.W., Stewart J.C., Villasmil M.L. Changes in fluorescence intensity of selected leukocyte surface markers following fixation. Cytometry A 2007, 71A:379-385.
Franck T., Grulke S., Deby-Dupont G., Deby C., Duvivier H., Peters F., Serteyn D. Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood. J. Vet. Diagn. Invest. 2005, 17:412-419.
Franck T., Kohnen S., Deby-Dupont G., Grulke S., Deby C., Serteyn D. A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests. J. Vet. Diagn. Invest. 2006, 18:326-334.
Gray E., Thomas T.L., Betmouni S., Scolding N., Love S. Elevated activity and microglial expression of myeloperoxidase in demyelinated cerebral cortex in multiple sclerosis. Brain Pathol. 2008, 18:86-95.
Grulke S., Benbarek H., Caudron I., Deby-Dupont G., Mathy-Hartert M., Farnir F., Deby C., Lamy M., Serteyn D. Plasma myeloperoxidase level and polymorphonuclear leukocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results. Can. J. Vet. Res. 1999, 63:142-147.
Grulke S., Franck T., Gangl M., Peters F., Salciccia A., Deby-Dupont G., Serteyn D. Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease. Can. J. Vet. Res. 2008, 72:37-42.
Hoy A., Leininger-Muller B., Kutter D., Siest G., Visvikis S. Growing significance of myeloperoxidase in non-infectious diseases. Clin. Chem. Lab. Med. 2002, 40:2-8.
Kalayci O., Moallem H., Fikrig S., Chice S., Michl J. Selective binding of a monoclonal antibody to aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes. Cytometry 2000, 39:260-265.
Kappelmayer J., Gratama J.W., Karaszi E., Menendez P., Ciudad J., Rivas R., Orfao A. Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols. J. Immunol. Methods 2000, 242:53-65.
Kingston J.K., Bayly W.M., Sellon D.C., Meyers K.M., Wardrop K.J. Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. Am. J. Vet. Res. 2002, 63:840-844.
Krutzik P.O., Nolan G.P. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 2003, 55A:61-70.
Lanza F., Latorraca A., Moretti S., Castagnari B., Ferrari L., Castoldi G. Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells. Cytometry 1997, 30:134-144.
Leong C.F., Kalaichelvi A.V., Cheong S.K., Hamidah N.H., Rahman J., Sivagengei K. Comparison of myeloperoxidase detection by flow cytometry using two different clones of monoclonal antibodies. Malays. J. Pathol. 2004, 26:111-116.
Mullane K.M., Read N., Salmon J.A., Moncada S. Role of leukocytes in acute myocardial infarction in anesthetized dogs: relationship to myocardial salvage by anti-inflammatory drugs. J. Pharmacol. Exp. Ther. 1984, 228:510-522.
Mullane K.M., Kraemer R., Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J. Pharmacol. Methods 1985, 14:157-167.
Schindhelm R.K., van der Zwan L.P., Teerlink T., Scheffer P.G. Myeloperoxidase: a useful biomarker for cardiovascular disease risk stratification?. Clin. Chem. 2009, 55:1462-1470.
Schmid I., Uittenbogaart C.H., Giorgi J.V. A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 1991, 12:279-285.
Xia Y., Zweier J.L. Measurement of myeloperoxidase in leukocyte-containing tissues. Anal. Biochem. 1997, 245:93-96.
