Isolation of heterophils from peripheral blood in hens and analysis of heterophil functions by flow cytometry - a methodological study

Abstract

The aim of this study was to modify the flow cytometric method, which is used for analyzing neutrophils, to analyse the functions of avian heterophils. The blood samples used in the experiments were obtained from hens in slaughterhouses. Blood samples were collected from 10 hens for each trial. Within the scope of the present study, trials were carried out regarding the amount of blood, cell suspension, dihydrorodamine-123 (DHR-123), phorbol-12- myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the storage duration of blood samples and incubation time. The results showed that 0.5-3 ml of blood could be used to detect heterophil functions, and it would be ideal to conduct analyses using fresh blood samples. In addition, the results showed that blood stored at +4 °C for up to 8 hours may be also used if necessary. In order to isolate the cells, centrifugation for 30 minutes is sufficient, and it is appropriate to use a 30µL cell suspension. 2µL of DHR-123 should be used as a chemical probe to measure heterophil functions. Excessive use of DHR-123 affected the heterophil functions negatively. In addition, it was observed that using 2µL of fMLP, which is used as an oxidative burst stimulant, and 2µL of PMA as a stimulant of chemotaxic activity, were sufficient. It was concluded that incubation at 41 °C for 5 minutes after stimulating the heterophils is also sufficient. We conclude that the methods established in this study could be used to isolate heterophils and to analyze them by flow cytometry. Therefore, this study would contribute to further research and clinical studies in poultry.