Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin

Cytokine. 2000 Nov;12(11):1712-6. doi: 10.1006/cyto.2000.0764.


Detected levels of IL-6, TNF-alpha and leptin may be affected by methods of storage, anticoagulant or repeated freezing-thawing. Blood samples from 22 healthy subjects were: (i) allowed to stand for 1, 2, 4 or 6 h prior to, or after separation, before freezing at -70 degrees C; (ii) taken into tubes with lithium heparin, sodium citrate, EDTA or no anticoagulant, separated and frozen; and (iii) separated, and plasma repeatedly freeze-thawed for up to six cycles prior to assay. Leptin was assayed by RIA, and IL-6 and TNF-alpha by high-sensitivity ELISA. (i) IL-6 and TNF-alpha levels were not altered significantly in separated samples but IL-6 declined by mean (SEM) 14.3% (3.7%) and TNF-alphaincreased by 9.6% (2.3%) in samples left unseparated for 4 h (P=0.003 and 0.002, respectively). Leptin remained unchanged. (ii) Serum and EDTA-plasma samples gave comparable results for all three cytokines, but levels in the other anticoagulant samples were highly variable. (iii) IL-6 and leptin levels were not altered by up to 6 cycles of freeze-thawing, but TNF-alpha increased by 17.0% (3.7%) after 3 cycles. Concentrations of these molecules are significantly altered by storage conditions, therefore they need to be standardized for epidemiological and clinical studies, and between-study comparisons of levels may not be reliable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Anticoagulants / pharmacology
  • Blood Chemical Analysis / methods
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Freezing
  • Humans
  • Interleukin-6 / chemistry*
  • Leptin / chemistry*
  • Male
  • Middle Aged
  • Preservation, Biological
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Specimen Handling*
  • Time Factors
  • Tumor Necrosis Factor-alpha / chemistry*


  • Anticoagulants
  • Interleukin-6
  • Leptin
  • Tumor Necrosis Factor-alpha