Modulation of blood cell activation by four commonly used anticoagulants.

Author(s): Engstad CS, Gutteberg TJ, Osterud B

Publication: Thromb Haemost, 1997, Vol. 77, Page 690-6

PubMed ID: 9134644 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of anticoagulant on monocyte, platelet, and neutrophil activation in whole blood. LPS induction of TF activity, release of TNFalpha, release of lactoferrin, and secretion of PF4 were measured. PBMC were isolated and frozen at -20 degrees C prior to quantification of TF activity. Platelet poor plasma was frozen at -70 degrees C prior to analysis.

   Summary of Findings:

   Regardless of anticoagulant, TF activity increased as incubation time with LPS increased from 1 to 2 hours, peaking at 2 hours. TF activity decreased again after incubation with LPS of heparinized whole blood for 4 h or EDTA blood for 5 h. PBMC TF activity was significantly lower when EDTA or citrate was used as an anticoagulant rather than heparin, or hirudin. Regardless of anticoagulant, only LPS concentrations of 0.5 ng/mL significantly increased TF activity, and higher concentrations did not result in further increases in activity. As with TF activity, TNFalpha levels were affected by increasing incubation time with LPS. TNFalpha concentrations were significantly lower in platelet poor plasma from whole blood anticoagulated with EDTA when compared to citrate, heparin, or hirudin. TNFalpha concentrations increased when LPS concentrations were increased from 0.1 ng/mL to 5 ng/mL, but no further increases were seen with LPS concentrations of 10, 50, or 100 ng/mL, regardless of anticoagulant. Lactoferrin release was greatest in platelet poor plasma from hirudinized blood, followed by heparinized blood, then citrated blood, and least in EDTA blood. Platelet poor plasma from heparinized blood had significantly higher levels of PF4 than plasma from EDTA, citrated, Fragmin, or hirudinized blood. While incubation time at 37 degrees C had no effect on PF4 in EDTA or citrated blood, increasing incubation time increased plasma PF4 concentrations in heparinized, hirudinized, and Fragmin blood. LPS concentration had no significant effects on plasma PF4 concentration for any anticoagulant.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma
   • Cell - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Enzyme assay
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Peripheral blood mononuclear cells Platelet-poor plasma
   Biospecimen Acquisition	Anticoagulant	Citrate EDTA Fragmin Heparin Hirudin
   Analyte Extraction and Purification	Analyte isolation method	Saline incubation 0.05 ng/mL LPS 0.1 ng/mL LPS 0.5 ng/mL LPS 1.5 ng/mL LPS 10 ng/mL LPS 50 ng/mL LPS 100 ng/mL LPS
   Analyte Extraction and Purification	Incubation duration/condition	1 h with LPS 2 h with LPS 3 h with LPS 4 h with LPS 5 h with LPS 6 h with LPS 0 min at 37 degrees C 5 min at 37 degrees C 10 min at 37 degrees C 15 min at 37 degrees C 30 min at 37 degrees C 60 min at 37 degrees C 90 min at 37 degrees C

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