title: Are the cytokines interleukin-6 and angiogenin stable in frozen amniotic fluid?
abstract: OBJECTIVE
  To determine the stability of cytokines in frozen stored amniotic fluid samples, we measured angiogenin, a potent inducer of neovascularization and interleukin-6, an inflammatory cytokine, in the same sample of midtrimester amniotic fluid 1 year apart.
  
  
  METHODS
  In this study paired aliquots of amniotic fluid kept at -70 degrees C were immunoassayed for angiogenin and interleukin-6 at two different time periods 1 year apart. Inclusion criteria were (1) samples with clearly identifiable numbers, (2) no evidence of breaks in sealing of samples, and (3) singleton gestation. Amniotic fluid was immunoassayed for angiogenin and interleukin-6 in July 1995 and 1996. Angiogenin sensitivities were 0.078 and 0.026 ng/ml, interassay coefficients of variation were 3.8% and 4.6%, and intraassay coefficients of variation were 2.7% and 2.9%, respectively, in 1995 and 1996. Interleukin-6 sensitivities were 1.74 and 2.37 pg/ml, interassay coefficients of variation were 8.9% and 2.6%, and intraassay coefficients of variation were 3.5% and 1.9%, respectively, in 1995 and 1996. Statistical analysis included paired t test, Wilcoxon signed-rank test, and regression with p < 0.05 significant. Angiogenin and interleukin-6 values were normalized with natural log transformation for statistical analysis.
  
  
  RESULTS
  Paired amniotic fluid samples from 30 patients were immunoassayed from 1993 to 1995. The values of angiogenin were significantly lower in the 1996 assay compared with the 1995 assay (median 12.4 [range 5.6 to 61.3] vs 26.7 [range 13.6 to 159.2] ng/ml, p < 0.001). A significant correlation was found between the change in angiogenin levels and the year of the sample, with older samples having the greatest change in values (r=0.5, p=0.008). The values of interleukin-6 were significantly lower in the 1996 assay compared with the 1995 assay (median 230.8 [range 40.9 to 3711.3] vs 289.2 [range 53.7 to 19100.0] pg/ml, p < 0.001).
  
  
  CONCLUSIONS
  Angiogenin and interleukin-6 values in amniotic fluid appear to decrease with time despite optimal freezing conditions. The year of sampling and length of storage should be taken into consideration when evaluating amniotic fluid cytokine levels from stored samples.
authors.affiliation: Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: C Y
authors.initials: CY
authors.lastname: Spong
authors.forename: A
authors.initials: A
authors.lastname: Ghidini
authors.forename: M
authors.initials: M
authors.lastname: Ossandon
authors.forename: C N
authors.initials: CN
authors.lastname: Walker
authors.forename: J C
authors.initials: JC
authors.lastname: Pezzullo
mesh_terms: D000328:Adult; D000653:Amniotic Fluid / Q000737:chemistry*; D043925:Angiogenesis Inducing Agents; D004355:Drug Stability; D005260:Female; D005615:Freezing; D006801:Humans; D007118:Immunoassay; D015850:Interleukin-6 / Q000032:analysis*; D008297:Male; D011247:Pregnancy; D011506:Proteins / Q000032:analysis*; D012259:Ribonuclease, Pancreatic; D013997:Time Factors
chemical_list: D043925:Angiogenesis Inducing Agents; D015850:Interleukin-6; D011506:Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic