title: Impact of heparin and short term anesthesia on the quantification of cytokines in laboratory mouse plasma.
abstract: BACKGROUND
  Studies have reported that heparin may be unsuitable as an anticoagulant in human plasma samples when quantifying cytokines using multiplex bead array assays. For mouse samples, multiplex assays have been validated for serum and EDTA-plasma, but it remains to be elucidated whether heparin influences the quantification of cytokines, and if so - to what extent. Furthermore, laboratory mice are often anesthetized for blood sampling, which causes acute stress that may influence circulating cytokine concentrations and thus bias experimental results. The objectives of the present study were to identify whether specific cytokine concentrations varied between heparin-plasma, serum, and EDTA-plasma, and whether short isoflurane anesthesia would influence the concentrations of these cytokines in the circulation. Twenty-three acute phase and pro-inflammatory cytokines were quantified in matched serum, EDTA-plasma, and heparin-plasma samples from anesthetized and unanesthetized male NMRI mice using a multiplex assay. In addition, samples from unanesthetized mice were spiked with three levels of heparin.
  
  
  RESULTS
  The concentrations of five out of 23 cytokines were significantly different between sample types, but only one cytokine (IL-17A) differed between heparin-plasma and serum. When further spiking the heparin-plasma with increasing concentrations of heparin, there was a significant effect on 11 cytokines, where the cytokine recovery could be correlated to the heparin concentration for ten of these cytokines. Anesthesia resulted in lower concentrations of G-CSF, but had no significant impact on the concentrations of the other 22 cytokines.
  
  
  CONCLUSIONS
  In mice, heparin seems like a suitable anticoagulant for obtaining plasma for multiplex assays for the cytokines IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα, but an effect of heparin in high concentrations should be considered for the cytokines IL-9, IL-12p40, IL-12p70, KC, MCP-1, MIP-1β and RANTES. Short isoflurane anesthesia had significant impact on G-CSF, but none of the other cytokines.
authors.affiliation: Department of Experimental Medicine, University of Copenhagen, Blegdamsvej 3B, Copenhagen DK-2200, Denmark. annete@sund.ku.dk.
authors.forename: Anne C
authors.initials: AC
authors.lastname: Teilmann
authors.forename: Otto
authors.initials: O
authors.lastname: Kalliokoski
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Jacobsen
authors.forename: Jann
authors.initials: J
authors.lastname: Hau
authors.forename: Klas S P
authors.initials: KS
authors.lastname: Abelson
mesh_terms: D000758:Anesthesia; D000818:Animals; D021461:Animals, Outbred Strains; D001774:Blood Chemical Analysis; D016207:Cytokines / Q000097:blood*; D004492:Edetic Acid / Q000097:blood*; D006493:Heparin / Q000097:blood*; D008297:Male; D051379:Mice
chemical_list: D016207:Cytokines; D006493:Heparin; D004492:Edetic Acid