Objective
Amniotic fluid specimens are commonly frozen and stored for research purposes. A previous study examined the stability of certain cytokines in stored amniotic fluid samples for prolonged periods of time. They showed some degradation of the cytokines despite optimal freezing conditions. On the other hand, little is known about how preanalytical handling procedures affect cytokine levels. Since amniotic fluid interleukin (IL)-6 is the most reported biomarker to identify intraamniotic inflammation, the purpose of this study was to evaluate the effect of different handling procedures on immunoassay measurable concentrations of IL-6 in amniotic fluid.
Study Design
From June through August 2012, amniotic fluid was collected from 21 women between gestational weeks 38+0 and 41+6, undergoing elective cesarean delivery at Sahlgrenska University Hospital/Östra, Gothenburg, Sweden. The study was approved by the local ethical committee at the University of Gothenburg (Dnr 991-11). Samples were obtained before the complete hysterotomy was performed, and 45 mL of amniotic fluid was aspired. Each amniotic fluid sample was separated into 9 groups of 4.5 mL each. The standard group was defined as the group with centrifugation of the sample at 2000 g , for 10 minutes, in 4°C within 2 hours from sampling, without addition of protease inhibitor and without supernate filtration. This approach was the preanalytical procedure of the amniotic fluid that our research group has previously used. In the other groups (test groups), one variable was changed to evaluate the influence of latency of time from sampling to centrifugation (5 hours of storage in group 1 and 24 hours in group 2), the centrifugal force (300 g in group 3 and 12,000 g in group 4), centrifugation time (20 minutes in group 5), centrifugation temperature (20°C in group 6), supernate filtration (group 7), and serine-, cysteine-, and metalloproteases inhibitor (group 8). After processing, aliquots from each group were immediately stored at –80°C until analysis with enzyme-linked immunosorbent assay within 8 months from sampling. Each enzyme-linked immunosorbent assay performed was specific for IL-6 with a detection rate of 15.6-1500 pg/mL. The interassay and intraassay coefficient of variation was <10% and <5%, respectively. Results were calculated by pairing the concentrations of IL-6 in the standard group with those in each test group using Wilcoxon signed ranks test. A value of P < .05 was considered significant.
Study Design
From June through August 2012, amniotic fluid was collected from 21 women between gestational weeks 38+0 and 41+6, undergoing elective cesarean delivery at Sahlgrenska University Hospital/Östra, Gothenburg, Sweden. The study was approved by the local ethical committee at the University of Gothenburg (Dnr 991-11). Samples were obtained before the complete hysterotomy was performed, and 45 mL of amniotic fluid was aspired. Each amniotic fluid sample was separated into 9 groups of 4.5 mL each. The standard group was defined as the group with centrifugation of the sample at 2000 g , for 10 minutes, in 4°C within 2 hours from sampling, without addition of protease inhibitor and without supernate filtration. This approach was the preanalytical procedure of the amniotic fluid that our research group has previously used. In the other groups (test groups), one variable was changed to evaluate the influence of latency of time from sampling to centrifugation (5 hours of storage in group 1 and 24 hours in group 2), the centrifugal force (300 g in group 3 and 12,000 g in group 4), centrifugation time (20 minutes in group 5), centrifugation temperature (20°C in group 6), supernate filtration (group 7), and serine-, cysteine-, and metalloproteases inhibitor (group 8). After processing, aliquots from each group were immediately stored at –80°C until analysis with enzyme-linked immunosorbent assay within 8 months from sampling. Each enzyme-linked immunosorbent assay performed was specific for IL-6 with a detection rate of 15.6-1500 pg/mL. The interassay and intraassay coefficient of variation was <10% and <5%, respectively. Results were calculated by pairing the concentrations of IL-6 in the standard group with those in each test group using Wilcoxon signed ranks test. A value of P < .05 was considered significant.
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