Application of flow cytometry and microscopy methods in erythrophagocytosis measurement

Abstract

Background: Phagocytic cells engulf erythrocytes coated with IgG antibodies and/or C3 complement components in specific cellular response. Since interaction of antibody-coated RBCs with phagocytes is evaluated mainly by measurements of the phagocytic index using a subjective microscopy method, we have applied the flow cytometry technique for erythrophagocytosis research. Aim of the study: To evaluate and compare cytometric and microscopic methods in detection and quantification of cells involved in erythrophagocytosis. Material and methods: Monocytes and erythrocytes were isolated from the 52 human blood samples. RhD positive donor's RBCs were opsonized by specific human IgG anti-D diagnostic serum. Flow cytometry measurements of CD14+ monocytes and CD235+ erythrocytes incubated at 37°C or 4°C were performed using FACSCantoII cytometer and FACSDiva Software. Quantification of the erythrophagocytosis process was performed using cells fixed on a microscopic slide, and dynamics of this process was carried out with the use of Nikon Diaphot 300 optical microscope and Nis Elements Software. Results: Statistical analysis of the results obtained by flow cytometry showed a lower coefficient of variation, and thus a more accurate measurement compared with the microscopic method. The flow cytometry procedure makes it possible to distinguish fully-ingested erythrocytes from these attached to the phagocyte, and hence to obtain more reliable results. Conclusions: Flow cytometry can be an alternative technique in detection and evaluation of red blood cells phagocytosis since it is relatively rapid, more sensitive and objective than microscopy.