New method to quantify erythrophagocytosis by autologous monocytes.
BACKGROUND
Anemia is the net result of decreased red blood cell (RBC) production and increased removal of RBCs. Replication and maturation of erythroid precursors and RBC lysis can be measured by standardized in vitro methods and surrogate markers, respectively. In contrast, erythrophagocytosis by autologous phagocytes is more difficult to quantify.


METHODS
We developed a method to assess erythrophagocytosis by autologous monocytes from 5 ml of whole blood. RBCs were labeled with carboxyfluorescein-diacetate-succinimidyl ester (CFDA-SE) and subsequently coincubated with autologous CD14(+) monocytes. Phagocytosis was quantified using flow cytometry. After standardization, the assay was validated in patients with severe malarial anemia (SMA), a condition that is associated with increased erythrophagocytosis.


RESULTS
After labeling, CFDA-SE was stably incorporated into RBCs and no significant leakage leading to contamination of nonlabeled cells was observed. Monocytes ingested opsonized, labeled RBCs seven times more than nonopsonized controls. Erythrophagocytosis was significantly higher in SMA than in healthy controls.


CONCLUSIONS
The established assay showed enhanced autoerythrophagocytosis associated with SMA and hence was able to detect clinically relevant erythrophagocytosis. This novel assay is well suited for rapid quantification of in vitro erythrophagocytosis by autologous monocytes.
Fendel
Rolf
R
Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon.
Mordmüller
Benjamin
B
Kreidenweiss
Andrea
A
Rudat
Annika
A
Steur
Claudia
C
Ambrosch
Christian
C
Kirstein
Max
M
Berdel
Wolfgang E
WE
Kremsner
Peter G
PG
Brandts
Christian
C
D000740:Anemia / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008856:Microscopy, Fluorescence / Q000379:methods*; D009000:Monocytes / Q000378:metabolism*; D010587:Phagocytosis; D013388:Succinimides / Q000378:metabolism
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D013388:Succinimides