Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions.

Author(s): Thavasu PW, Longhurst S, Joel SP, Slevin ML, Balkwill FR

Publication: J Immunol Methods, 1992, Vol. 153, Page 115-24

PubMed ID: 1381403 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of collection tube, anticoagulant, and storage at 4 degrees C or RT on cytokines in blood and plasma specimens. Specimens were spiked with human recombinant cytokines unless otherwise stated.

   Summary of Findings:

   Cytokine levels in unspiked blood were below the reliable levels of detection, and no evidence of cytokine induction was found. TNF-alpha and IL-6 decreased with time at 4 degrees C (p<0.001) or RT (p<0.001), but the decrease in IL-6 was less pronounced at 4 degrees C. Similarly, IFN-gamma decreased with time at 4 degrees C or RT in EDTA or serum specimens (p<0.0001), but not in heparinized specimens where IFN-gamma recovery was twice as high despite having been similarly spiked. IL-1a and IFN-alpha decreased with time at RT (p<0.001) but not at 4 degrees C. In contrast, IL-1b levels did not change with storage at either temperature in any tube type. No differences in TNF-alpha or IL-1a recovery rates were observed with different anticoagulants or with the addition of protease inhibitors to EDTA, but IL-6 recovery was consistently higher when EDTA was used. TNF-alpha, IL-1a, and IL-6 recovery rates were lower in serum. Use of commercial reagents instead of sterile non-pyrogenic materials gave similar results for all cytokines except IFN-gamma where the effects of time and temperature were lost.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Radioimmunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	EDTA Heparin None
   Biospecimen Acquisition	Type of collection container/solution	Sterile non-pyrogenic material and conditions Commercial tubes
   Storage	Storage temperature	4 degrees C Room temperature -40 degrees C
   Storage	Storage duration	15 min 30 min 1 h 2 h 4 h 24 h
   Biospecimen Aliquots and Components	Biospecimen components	Spiked with cytokine Not spiked
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Analyte Extraction and Purification	Protease inhibitor	Aprotinin No protease inhibitor added

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2. Study Purpose

   The purpose of this study was to evaluate the effects of anticoagulant and up to 6 freeze-thaw cycles at 4 degrees C or RT on cytokine levels. All specimens in this study were spiked with human recombinant cytokines.

   Summary of Findings:

   No obvious differences were observed between specimens thawed at 4 degrees C and those thawed at room temperature. TNF-alpha, IL-1a, IL-1b and IFN-gamma were stable through 6 freeze-thaw cycles regardless of anticoagulant used, however TNF-alpha was significantly lower in serum specimens and IFN gamma was significantly higher in heparinized plasma. IL-6 was stable for at least three freeze-thaw cycles, but was very inconsistent in heparinized plasma.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Radioimmunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Heparin EDTA None
   Storage	Storage temperature	4 degrees C Room temperature -40 degrees C
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum

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