Counting absolute numbers of specific leukocyte subpopulations in avian whole blood using a single-step flow cytometric technique: comparison of two inbred lines of chickens

A novel flow cytometric technique was developed to determine the absolute numbers of leukocytes of specific phenotypes in whole blood from two lines of inbred chickens (line 7 2 and line 6 1). This single step method is rapid, accurate, repeatable, can be used in the presence of nucleated erythrocytes and addresses the problems encountered when electronically counting the numbers of leukocytes in specific subpopulations in the blood of non-mammalian species. It is superior to previous methods in that (1) peripheral blood leukocytes (PBL) do not need to be separated by density gradient centrifugation, (2) erythrocyte lysis is not necessary and (3) absolute numbers of specific phenotypes of cells are determined directly. A standard volume of diluted whole blood was added to a standard number of fluorescent beads before incubation with fluorescently-conjugated monoclonal antibodies recognising specific PBL surface antigens. Samples were analysed by flow cytometry and electronic gates were set to count a standard number of beads and the concomitant fluorescently-labelled cells. Absolute numbers of B, CD4 + and CD8 + PBL were determined. Since the bead fluorescence is constant, it was also possible to measure relative MHC class I expression using fluorescence intensity. In both lines of chickens absolute numbers of all of the phenotypes of PBL measured increased with age. Although line 7 2 chickens had greater numbers of B, CD4 +, and CD8 + PBL than line 6 1 chickens, there was no significant difference in the CD4 +:CD8 + PBL ratios, the T:B PBL ratios or relative MHC class I expression between the two lines. Relative MHC class I expression increased with age in both lines.