Figure 1
Figure 1. CD163/CD33 coexpression and decreased CD163 expression after treatment with gemtuzumab ozogamicin during intravascular hemolysis (patient 1). (A) Time-course of C-reactive protein (CRP) concentrations after GO-treatment in relation to episodes of infection (yellow area) and hemolysis (red area). (B) Coexpression of CD33 and CD163 on CD14 positive monocytes in healthy: Flow cytometric analysis of CD33 and CD163 expression on CD14 positive monocytes in freshly drawn EDTA-stabilized peripheral whole blood from a healthy individual (results are representative of 5 independent experiments). After gating of mononuclear cells (forward- vs side-scatter) and monocytes (CD14+), cells were replotted with CD33 FITC (FL1) versus CD163 (MAC2-158) PE (FL2). (C) Nonspecific FITC- and PE-conjugated IgG isotype-matched controls served as negative controls. (D) Double immunofluorescence microscopy revealed the simultaneous presence of CD33 and CD163 on Kupffer cells (top left panel: DAPI, blue immunofluorscence; top right panel: CD33, green immunofluorscence; bottom left panel: CD163, red immunofluorescence; bottom right panel: overlay, orange/yellow, marked with white arrowheads; original magnification ×100). (E) Immunofluorescence confocal laser scanning microscopy revealed a colocalization of CD33 and CD163 in bone marrow sections from patient 1 (relapse of acute myeloid leukemia before GO-treatment; left panel: FITC-conjugated anti-CD33, green immunofluorscence; middle panel: biotin-conjugated anti-CD163/streptavidin-conjugated Alexa-Fluor 594, red immunofluorescence; right panel: overlay, yellow, marked with white arrowheads; original magnification ×63). (F,G) Flow cytometric analysis of CD163 expression on peripheral blood cells during the hemolysis episode in patient 1 (F), compared with a healthy individual (G). CD163 positive cells were stained with PE (FL2), showing an almost complete depletion of CD163 positive cells (F; right panel). The results shown here from patient 1 are completely consistent with the data for patient 2, which are shown in Figure S1.

CD163/CD33 coexpression and decreased CD163 expression after treatment with gemtuzumab ozogamicin during intravascular hemolysis (patient 1). (A) Time-course of C-reactive protein (CRP) concentrations after GO-treatment in relation to episodes of infection (yellow area) and hemolysis (red area). (B) Coexpression of CD33 and CD163 on CD14 positive monocytes in healthy: Flow cytometric analysis of CD33 and CD163 expression on CD14 positive monocytes in freshly drawn EDTA-stabilized peripheral whole blood from a healthy individual (results are representative of 5 independent experiments). After gating of mononuclear cells (forward- vs side-scatter) and monocytes (CD14+), cells were replotted with CD33 FITC (FL1) versus CD163 (MAC2-158) PE (FL2). (C) Nonspecific FITC- and PE-conjugated IgG isotype-matched controls served as negative controls. (D) Double immunofluorescence microscopy revealed the simultaneous presence of CD33 and CD163 on Kupffer cells (top left panel: DAPI, blue immunofluorscence; top right panel: CD33, green immunofluorscence; bottom left panel: CD163, red immunofluorescence; bottom right panel: overlay, orange/yellow, marked with white arrowheads; original magnification ×100). (E) Immunofluorescence confocal laser scanning microscopy revealed a colocalization of CD33 and CD163 in bone marrow sections from patient 1 (relapse of acute myeloid leukemia before GO-treatment; left panel: FITC-conjugated anti-CD33, green immunofluorscence; middle panel: biotin-conjugated anti-CD163/streptavidin-conjugated Alexa-Fluor 594, red immunofluorescence; right panel: overlay, yellow, marked with white arrowheads; original magnification ×63). (F,G) Flow cytometric analysis of CD163 expression on peripheral blood cells during the hemolysis episode in patient 1 (F), compared with a healthy individual (G). CD163 positive cells were stained with PE (FL2), showing an almost complete depletion of CD163 positive cells (F; right panel). The results shown here from patient 1 are completely consistent with the data for patient 2, which are shown in Figure S1.

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