Ex vivo induction of cytokine mRNA expression in human blood samples.

Author(s): Härtel C, Bein G, Müller-Steinhardt M, Klüter H

Publication: J Immunol Methods, 2001, Vol. 249, Page 63-71

PubMed ID: 11226464 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 blood withdrawal speed (0.4 mL/s versus 2 mL/s) and isolation of PBMC as opposed to isolation of leukocytes from whole blood on cytokine mRNA levels. The effects of culturing whole blood or isolated PBMC for 24 h prior to isolation of RNA were also examined. Accelerated blood flow was achieved by rapid withdrawal of the collection tube piston during collection to create a vacuum effect.

   Summary of Findings:

   When cytokine mRNA was quantified in leukocytes from fresh whole blood, specimens obtained with accelerated blood flow had higher expression of IL-2, IL-4, and TNF-alpha (P=0.018, P=0.028, and P=0.018, respectively) than specimens obtained under moderate blood flow. Specimens from 5 of 8 donors also showed higher IFN-gamma expression when accelerated rather than moderate blood flow was employed, but this difference was not significant. There were no significant differences between native whole blood and unstimulated cultured whole blood in the levels of cytokine mRNA for either withdrawal rate. Cytokine mRNA quantified in PBMC also revealed upregulation of IL-2, IL-4, and TNF-alpha compared to levels measured in isolated leukocytes from fresh whole blood (moderate blood flow specimens), and PBMC cultures showed the same upregulation in these three transcripts. Specimens from 3 of 8 donors also showed higher IFN-gamma mRNA levels in PBMC than in leukocytes from fresh whole blood, but this difference was not significant. Isolation of PBMC caused 5-9-fold increases in IL-2, IL-4, and TNF-alpha mRNA expression while accelerated blood flow during specimen collection caused 1.8-2.6-fold increases in the same transcripts.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells
   • Cell - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of fluid acquisition	Blood flow rates compared
   Biospecimen Aliquots and Components	Blood and blood products	Leukocyte Peripheral blood mononuclear cells Whole blood Cultured peripheral blood mononuclear cells Unstimulated cultured whole blood
   Real-time qRT-PCR Specific	Targeted nucleic acid	IL-2 IL-4 TNF-alpha IFN-gamma

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