Influence of EDTA and Heparin on Lipopolysaccharide Binding and Cell Activation, Evaluated at Single-Cell Level in Whole Blood

Cytometry. 2002 Feb 15;50(1):14-8.

Abstract

Background: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed.

Methods: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha).

Results: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood.

Conclusion: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anticoagulants / pharmacology
  • Antigens, CD / biosynthesis
  • Antigens, Differentiation, T-Lymphocyte / biosynthesis
  • Dose-Response Relationship, Immunologic
  • Edetic Acid / pharmacology*
  • Flow Cytometry
  • Heparin / pharmacology*
  • Humans
  • Lectins, C-Type
  • Lipopolysaccharides / metabolism*
  • Lymphocyte Activation / drug effects*
  • Monocytes / drug effects
  • Monocytes / metabolism
  • Protein Binding / drug effects
  • Salmonella / immunology
  • Tumor Necrosis Factor-alpha / biosynthesis

Substances

  • Anticoagulants
  • Antigens, CD
  • Antigens, Differentiation, T-Lymphocyte
  • CD69 antigen
  • Lectins, C-Type
  • Lipopolysaccharides
  • Tumor Necrosis Factor-alpha
  • Heparin
  • Edetic Acid