A high-sensitivity assay for amniotic fluid insulin at 14-20 weeks' gestation

To examine sensitivity, precision, and sample stability of an immunochemiluminomimetric insulin assay in 14-20 week amniotic fluid (AF) and fetal age distribution of second-trimester AF insulin concentrations.

We assayed fresh specimens from 576 gravidas who had amniocentesis at 14-20 weeks' gestation because of maternal age. In a preliminary study, samples were divided into aliquots and stored at 4C and -20C for 24 hours to assess freezing effect. Some samples stored at 4C were assayed repeatedly during a 14-day period and others, stored at -20C, were assayed after a 70-day period.

This assay reliably measured AF insulin to a detection limit of 0.03 microIU/mL. Insulin could be measured in all amniotic fluid samples and demonstrated a log10 Gaussian distribution, ranging from 0.24 to 7.41 microIU/mL. Interassay coefficients of variation ranged from 4.4 to 8.9% at concentrations of 0.4-2.0 microIU/mL. Linearity of dilution from 1.5 to 10 times was 99.2 +/- 8.6%. Spike recovery of 10 microIU/mL was from 92-109%. Recovery after freezing to -20C for 24 hours (101%) and 70 days (97%) and after storage at 4C for 14 days (97%) demonstrated no significant loss.

A two-site, dual monoclonal, immunochemiluminomimetric insulin assay was sufficiently sensitive and precise within the lower range of measured AF insulin concentrations to investigate clinical associations of 14-20 week AF insulin with maternal and fetal conditions. The insulin stability in this matrix suggests that assays can be reliable on specimens stored up to 70 days.