eISSN: 1644-4124
ISSN: 1426-3912
Central European Journal of Immunology
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2/2013
vol. 38
 
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abstract:

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

Anna Stachurska
,
Katarzyna Gmerek
,
Jadwiga Fabijańska-Mitek

(Centr Eur J Immunol 2013; 38 (2): 208-213)
DOI: https://doi.org/10.5114/ceji.2013.35217
Online publish date: 2013/07/08
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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.
keywords:

erythrophagocytosis, RBCs opsonization, CD235a labelling, CD14 labelling
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