title: Optimization of cytokine stability in stored amniotic fluid.
abstract: OBJECTIVE
  Many studies use stored amniotic fluid samples to assay cytokines and other proteins for outcome-based research; however, there is little information on the optimal methods of storage. The objective of our study was to evaluate cytokine stability in amniotic fluid stored at different temperatures both with and without a proteolytic enzyme inhibitor.
  
  
  METHODS
  Patients undergoing midtrimester genetic amniocentesis for routine indications gave consent for the study. After the sample was centrifuged, the acellular portion of the sample was mixed to homogeneity and aliquoted in 0.5-mL increments and stored for 1 year at 4 degrees C, -20 degrees C, and -80 degrees C with and without the protease inhibitor aprotinin. Enzyme-linked immunoassays for angiogenin, interleukin-6, and vascular endothelial growth factor were performed simultaneously on each aliquot.
  
  
  RESULTS
  Thirty samples were assayed for each storage condition. Results were calculated as the percentage of its own sister aliquot stored at -80 degrees C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4 degrees C and the highest at -80 degrees C (angiogenin, P =.004; interleukin-6, P <.001; vascular endothelial growth factor, P =.02). The addition of aprotinin improved stability only for angiogenin at all temperatures (all P <.05).
  
  
  CONCLUSIONS
  Degradation of cytokines occurs when amniotic fluid samples are stored for prolonged periods at temperatures greater than -80 degrees C. The addition of a protease inhibitor helps stem the degradation of some cytokines.
authors.affiliation: Department of Obstetrics and Gynecology, Georgetown University, Washington, DC, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Porter
authors.forename: J
authors.initials: J
authors.lastname: Auth
authors.forename: M
authors.initials: M
authors.lastname: Prince
authors.forename: A
authors.initials: A
authors.lastname: Ghidini
authors.forename: D E
authors.initials: DE
authors.lastname: Brenneman
authors.forename: C Y
authors.initials: CY
authors.lastname: Spong
mesh_terms: D000653:Amniotic Fluid / Q000737:chemistry*; D007611:Aprotinin; D015925:Cryopreservation; D016207:Cytokines / Q000032:analysis*; D004355:Drug Stability; D016228:Endothelial Growth Factors / Q000032:analysis; D005260:Female; D005865:Gestational Age; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis; D007621:Karyotyping; D008222:Lymphokines / Q000032:analysis; D011247:Pregnancy; D011446:Prospective Studies; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D013696:Temperature; D013997:Time Factors; D014021:Tissue Preservation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: D016207:Cytokines; D016228:Endothelial Growth Factors; D015850:Interleukin-6; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000509:alpha-Fetoproteins; D007611:Aprotinin; C046366:angiogenin; D012259:Ribonuclease, Pancreatic