title: Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin.
abstract: 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.
authors.affiliation: Department of Medicine, Royal Free and University College Medical School, University College London, Whittington Campus, London, UK. rmhalof@ucl.ac.uk
authors.forename: L
authors.initials: L
authors.lastname: Flower
authors.forename: R H
authors.initials: RH
authors.lastname: Ahuja
authors.forename: S E
authors.initials: SE
authors.lastname: Humphries
authors.forename: V
authors.initials: V
authors.lastname: Mohamed-Ali
mesh_terms: D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D001774:Blood Chemical Analysis / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005615:Freezing; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry*; D020738:Leptin / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D011309:Preservation, Biological; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
chemical_list: D000925:Anticoagulants; D015850:Interleukin-6; D020738:Leptin; D014409:Tumor Necrosis Factor-alpha