title: The influence of different anticoagulants and sample preparation methods on measurement of mCD14 on bovine monocytes and polymorphonuclear neutrophil leukocytes.
abstract: BACKGROUND
  Membrane-CD14 (mCD14) is expressed on the surface of monocytes, macrophages and polymorphonuclear neutrophil leukocytes (PMN). mCD14 acts as a co-receptor along with Toll like receptor 4 (TLR 4) and MD-2 for the detection of lipopolysaccharide (LPS). However, studies using different sample preparation methods and anticoagulants have reported different levels of mCD14 on the surface of monocytes and neutrophils. In this study, the influence of various anticoagulants and processing methods on measurement of mCD14 on monocytes and neutrophils was examined.
  
  
  RESULTS
  Whole blood samples were collected in vacutainer tubes containing either sodium heparin (HEPARIN), ethylenediaminetetraacetic acid (EDTA) or sodium citrate (CITRATE). mCD14 on neutrophils and monocytes in whole blood samples or isolated cells was measured by the method of flow cytometry using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. There was a significant difference (p < 0.05) in the mean channel fluorescence intensity (MFI) of mCD14 on neutrophils in whole blood samples anticoagulated with HEPARIN (MFI = 64.77) in comparison with those in whole blood samples anticoagulated with either EDTA (MFI = 38.25) or CITRATE (MFI = 43.7). The MFI of mCD14 on monocytes in whole blood samples anticoagulted with HEPARIN (MFI = 206.90) was significantly higher than the MFI in whole blood samples anticoagulated with EDTA (MFI = 149.37) but similar to that with CITRATE (MFI = 162.55). There was no significant difference in the percentage of whole blood neutrophils or monocytes expressing mCD14 irrespective of type of anticoagulant used. However, MFI of mCD14 on monocytes was about 3.2-folds (HEPARIN), 3.9-folds (EDTA) or 3.7 folds (CITRATE) higher than those on neutrophils. Furthermore, there was no significant difference in mCD14 levels between unprocessed whole blood monocytes and monocytes in peripheral blood mononuclear cell preparation. Conversely, a highly significant difference was observed in mCD14 between unprocessed whole blood neutrophils and isolated neutrophils (p < 0.05).
  
  
  CONCLUSIONS
  From these results, it is suggested that sodium heparin should be the preferred anticoagulant for use in the reliable quantification of the surface expression of mCD14. Furthermore, measurement of mCD14 is best carried out in whole blood samples, both for neutrophils and monocytes.
authors.affiliation: Department of Animal Science, McGill University, Quebec, Canada.
authors.forename: Eveline M
authors.initials: EM
authors.lastname: Ibeagha-Awemu
authors.forename: Aloysius E
authors.initials: AE
authors.lastname: Ibeagha
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002951:Citrates / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D006493:Heparin / Q000737:chemistry*; D018950:Lipopolysaccharide Receptors / Q000032:analysis*; D009000:Monocytes / Q000737:chemistry* / Q000166:cytology; D009504:Neutrophils / Q000737:chemistry* / Q000166:cytology; D000077559:Sodium Citrate; D013048:Specimen Handling / Q000379:methods*
chemical_list: D000925:Anticoagulants; D002951:Citrates; D018950:Lipopolysaccharide Receptors; D000077559:Sodium Citrate; D006493:Heparin; D004492:Edetic Acid