14 ng/ml. Samples were diluted 14 using phosphate buffer saline. Absorbance values were read at 450 nm using Multiskan RC ELISA reader (Thermo Fisher Scientific, Waltham, MA). Statistical Analyses of ELISA Data from Validation and Replication Cohorts The demographic and clinical characteristics were compared Nutlin 3a using unpaired t-tests for continuous variables (presented as the mean �� SD) or the Mann-Whitney U test for nonparametric variables (presented as the median along with the range). Categorical variables were compared using the Fisher exact test and are presented as number (%). The normality of the data was tested using the D��Agostino and Pearson omnibus normality test and the Shapiro-Wilk test.

Because concentrations of cathelicidin were not normally distributed, the nonparametric Mann-Whitney U test was used for analyses and data are presented as median [interquartile range (IQR)]. Receiver-operator characteristic (ROC) curves were constructed to determine the predictive value of cathelicidin for the presence of both MIAC and HCA. Cut-off point of amniotic fluid cathelicidin was chosen based on the maximum likelihood ratio (LR) calculated from exploratory cohort cathelicidin levels. Differences were considered statistically significant at p<0.05. All p-values were obtained from two-sided tests. All statistical analyses were performed using GraphPad Prism 5.03 for Mac OS X (GraphPad Software, La Jolla, CA), SPSS 19.0 statistical package for Mac OS X (SPSS Inc., Chicago, IL), and PASS 11 (NCSS, Kaysville, UT).

Results Exploratory Phase of the Study Demographic and clinical characteristics of the exploratory cohort For the initial exploratory phase of the study we employed 19 amniotic fluid samples in each group to be compared. Table 1 presents the demographic and clinical characteristics of both women and newborns according to the presence and the absence of MIAC and HCA. All women were self-reported as Caucasians. Table 1 Demographic and clinical characteristics of women and newborns involved in the exploratory cohort. Exploratory proteomic analysis The exploratory proteomic study of pooled amniotic fluid samples obtained from the exploratory cohort patients involved removal of 14 ballast proteins, peptide fractionation based on the presence of cysteine residues, initial separation on reversed-phase in basic conditions, and eventually reversed-phase HPLC-MALDI-TOF/TOF analysis (Fig.

S1). This multidimensional nature of the study led to the recording of 20.382 MS/MS Dacomitinib spectra, identifying 9.422 distinct peptides at a maximum of 5% FDR [25]. Based on these peptides, 851 amniotic fluid proteins were successfully identified (5% FDR). Of these, 99 proteins were significantly (p��0.01) altered in both replicates (see Fig. 1 and Table S1 and S2).