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Jolanta Spuzak, Anna Chelmonska-Soyta*, Krzysztof Kubiak, Marcin Jankowski, Jozef Nicpon, Joanna Blach*, Urszula Paslawska, Kamila Glinska-Suchocka ®
Department of Internal and Parasitic Diseases with Clinic for Horses, Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life
Sciences, pl. Grunwaldzki 47, 50-366 Wroclaw, Poland *Department and Clinic of Obsterics, Ruminant Diseases and Animal Health, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, pl.
Grunwaldzki 49, 50-366 Wroclaw, Poland
APPLICATION OF FLOW CYTOMETRY IN BLOOD EXAMINATION AND BRONCHOALVEOLAR LAVAGE IN HEALTHY DOGS
Key words: flow cytometry, BAL, blood, dog
Introduction
Flow cytometry is a technique allowing a multiparameter evaluation of the structure and function of cells. It allows, among others, cells immunophenotyping, evaluation of DNA and RNA content in cells, evaluation of phagocytosing cells activity and evaluation of cytotoxicity in relation to target cells (1, 2, 3, 4).
Task, the aim of the article
The aim of the study was to determine the number of cells contained in of bronchoalveolar lavage in healthy dogs and the percentage composition of single cell populations. Another aim of the study was to determine differences in size of T CD4+ and T CD8+ lymphocytes subpopulations and the ratio of CD4/CD8+ lymphocytes isolated from blood and bronchoalveolar lavage in healthy dogs.
Material and methods
The research was conducted on 8 dogs, of different breed, size and sex, aged 1-8 years. Blood and bronchoalveolar lavage were collected from all the dogs. Branchoalveolar lavage was collected during bronchoscopy which was carried out under general anesthesia after 24-hour fasting and a 6-hour break in water administration. Premedication comprised: xylasine - 1-2 mg/kg b.w. with atropine -0.05 mg/kg b.w, whereas for general anesthesia tiopental was used - in the initial dose 5 mg/kg b.w., and next according to the effect of action.. For local anesthesia of the throat and bronchus area 1% lignocaine was used. The number of cells contained in 1 ^l of lavage was calculated in Thom chamber. A part of lavage was centrifuged to obtain sediment from which cytological specimens were made. In the specimens the percentage composition of single cell populations was determined. The blood in tubes with heparin and a part of lavage were referred for examinations with the use of a flow cytometer and the size of T CD4+ and T CD8+ lymphocytes subpopulations as
® Jolanta Spuzak, Anna Chelmonska-Soyta, Krzysztof Kubiak, Marcin Jankowski, Jozef Nicpon, Joanna Blach, Urszula Paslawska, Kamila Glinska-Suchocka, 2008
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well as CD4/CD8+ ratio were determined. RAT ANTI CANINE CD45R:FITC monoclonal antibodies were used for isolation of leucocytes, ANTI CANINE CD3:FITC/CD4:RPE antibodies were used for isolation of T CD4+ lymphocytes and ANTI CANINE CD3:FITC/CD8:RPE antibodies (Serotec company) were used for isolation of T CD8+ lymphocytes. Blood and bronchoalveolar lavage samples were prepared according to methodics recommended by the producer.
Results of research
In bronchoalveolar lavage on average 285 ± 113,14 cells were obtained in 1^l. On the basis of the cytological examination it was found that alveolar macrophages, which constituted on average 59.75 ± 6.20%, were the dominant population. In lower quantities occurred lymphocytes - 27.81 ± 5.14%, neutrophils -6.25 ± 2.35%, eosinophils - 5.56 ± 1.99% and mast cells - 0.63 ± 1.77%.
In blood of the examined dogs T CD4+ lymphocytes constituted on average 44.89 ± 7.07%, T CD8+ lymphocytes - on average 28.59 ± 4.69% (Tab.1), whereas CD4/CD8+ ratio was on average 1.65 ± 0.62.
Tab.1. Size of T CD4+ and T CD8+ lymphocytes subpopulations in healthy
dogs'blood
In bronchoalveolar lavage T CD4+ lymphocytes constituted on average 19.86 ± 9.25%, T CD8+ - on average 39.67 ± 17.24% (Tab.2), whereas CD4/CD8+ ratio was on average 0.56 ± 0.31.
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Tab.2. Size of T CD4+ and T CD8+ lymphocytes subpopulations in healthy
dogs'bronchoalveolar lavage
Conclusions
The conducted research led to the conclusion that the dominant cell population in bronchoalveolar lavage are alveolar macrophages; the remaining cell populations occur in lower quantities. In the blood of healthy dogs a predominance of T CD4+ lymphocytes over T CD8+ lymphocytes was observed, and conversely - a predominance of T CD8+ lymphocytes over T CD4+ lymphocytes was observed in bronchoalveolar lavage.
Reference
1. Chlap Z.: Atlas cytopatologii chorob srodmi^zszowych pluc. Plukanie oskrzelowo-pçcherzykowe (BAL). Medycyna Praktyczna, 2001, program multimedialny.
2. Dirscherl P., Beisker W., Kremmer E., Mihalkov A., Voss C., Ziesenis A.: Immunophenotyping of canine bronchoalveolar and peripheral blood lymphocytes. Veterinary Immunology and Immunopathology, 1995, 48, 1-10.
3. Winnicka A.: Zastosowanie cytometrii przeplywowej w diagnostyce laboratoryjnej. Magazyn Weterynaryjny, 2005, 14, 4-6.
4. Vail D.M., Mahler P.A., Soergel S.A.: Differential cell analysis and phenotypic subtyping of lymphocytes in bronchoalveolar lavage fluid from clinically normal dogs. Am. J. Vet. Res., 1995, 56, 282-285.
Summary
The aim of the study was to determine the number of cells contained in 1^of bronchoalveolar lavage in healthy dogs and the percentage composition of single cell populations. Another aim of the study was to determine differences in size of T CD4+ and T CD8+ lymphocytes subpopulations and the ratio of CD4+/CD8+ lymphocytes isolated from blood and bronchoalveolar lavage in healthy dogs.
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The research was conducted on 8 dogs, of different breed, size and sex, aged 1-8 years. Blood and bronchoalveolar lavage were collected from all the dogs.
The conducted research led to the conclusion that the dominant cell population in bronchoalveolar lavage are alveolar macrophages; the remaining cell populations occur in lower quantities. In the blood of healthy dogs a predominance of T CD4+ lymphocytes over T CD8+ lymphocytes was observed, and conversely - a predominance of T CD8+ lymphocytes over T CD4+ lymphocytes was observed in bronchoalveolar lavage.
Стаття надшшла до редакцИ' 10.09.2008
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