Kinetic titration method in flow cytometry for quantification of cell receptors

Abstract

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

Keywords: Atherosclerosis; CD14; CD8; Dynamic flow cytometry; Leukocytes; Ligand-receptor binding kinetics; Mathematical model; Quantitative immunoassay; Reaction rate constants.