Optimization of cytokine stability in stored amniotic fluid

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. STUDY DESIGN: 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°C, -20°C, and -80°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°C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4°C and the highest at -80°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°C. The addition of a protease inhibitor helps stem the degradation of some cytokines.