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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.

Experimental immunology Application of flow cytometry and microscopy methods in erythrophagocytosis measurement