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CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli.