title: New method to quantify erythrophagocytosis by autologous monocytes.
abstract: 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.
authors.affiliation: Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mordmüller
authors.forename: Andrea
authors.initials: A
authors.lastname: Kreidenweiss
authors.forename: Annika
authors.initials: A
authors.lastname: Rudat
authors.forename: Claudia
authors.initials: C
authors.lastname: Steur
authors.forename: Christian
authors.initials: C
authors.lastname: Ambrosch
authors.forename: Max
authors.initials: M
authors.lastname: Kirstein
authors.forename: Wolfgang E
authors.initials: WE
authors.lastname: Berdel
authors.forename: Peter G
authors.initials: PG
authors.lastname: Kremsner
authors.forename: Christian
authors.initials: C
authors.lastname: Brandts
mesh_terms: 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
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D013388:Succinimides