Methods

Development cohort

We identified potentially eligible participants from the Oxford Pregnancy Biobank between April 2009 and April 2010 and examined them against our inclusion criteria to obtain an objectively healthy population of pregnant women from which to estimate RIs. The Oxford Pregnancy Biobank was a prospective study of pregnant women in Oxford, UK (REC 07/H0607/74), in which pregnancy-specific data were recorded in each trimester and after delivery. A maternal blood sample was taken and stored from the first, second and third trimesters at 10-14+6, 18-23+6, and 31-38+6 weeks, respectively.
The inclusion criteria for our study were adult pregnant women (≥18 years old) with uncomplicated pregnancies. We excluded women with chronic hypertension or diabetes mellitus, and women whose index pregnancy was complicated by gestational hypertension (blood pressure ≥140/90), pre-eclampsia (hypertension with proteinuria), HELLP syndrome (haemolysis, elevated liver enzymes, low platelets), acute fatty liver of pregnancy, gestational diabetes (abnormal oral glucose tolerance test) or obstetric cholestasis (pruritis with bile acids >14 µmol/L).

Analysis

Plasma CRP was measured using an immunoturbimetric method on the Abbott Architect C16000 analyser (Abbott Laboratories Ltd, Maidenhead, UK) after a single freeze-thaw cycle (-80°C). Inter-assay imprecision expressed as CV, % was 5.4% at 1.6 mg/L, and 1.0% at 53.6 mg/L, imprecision was 1.02%. The lower detection limit was 0.2 mg/L.

Statistics

Data on CRP in each trimester were approximated to a normal distribution using the natural logarithmic transformation:
\begin{equation} f\left(x\right)=ln(x-k)\nonumber \\ \end{equation}
where k was calculated to minimise skewness. Outliers were identified using Horn’s13 interpretation of the method described by Hoaglin, et al. 14 Data were binned into groups of maternal age and BMI, and participants were excluded in whom CRP was more extreme than 1.5 times the interquartile range above or below the 3rd or 1st quartiles, respectively. The transformations were reapplied after excluding outliers and Shapiro-Wilk tests were used to confirm the distributions.
RIs were calculated for each trimester, equating to the 2.5th and 97.5th percentiles derived from the mean and standard deviation, and the reference limits were presented with their respective 90% confidence intervals.15 One-way ANOVA or Student’s t-tests were used to investigate differences in CRP according to groups of maternal age, BMI, mean arterial pressure (at booking), ethnicity, and fetal sex. Participants with missing data in any variable were excluded from subgroup analyses as the frequency of missing data was minimal.16 We ran a multivariate linear regression model to investigate the association between CRP and BMI, and investigated partitioning the RIs using the method described by Lahti,et al .17 Summary statistics for normally distributed data are presented as the mean and standard deviation, or otherwise as frequencies and proportions. Statistical significance was assumed at the 5% level, or at the 1% level for subgroup analyses.

Evaluation cohort

We evaluated the diagnostic accuracy of the new upper reference limit for CRP for detecting infection, using chorioamnionitis as a well-defined example of infection. We retrospectively identified a subset of 50 consecutive women who had undergone amniocentesis for suspected infection, and extracted the maternal serum CRP to calculate the sensitivities, specificities, positive and negative predictive values, and areas under the receiving operator curves for infection using two reference limits:
The overall diagnostic accuracy of the two reference standards was compared using receiving operator curve (ROC) analysis. Chorioamnionitis was defined as microbiological growth or microscopic evidence of organisms in amniotic fluid, growth on a placental swab, and/or a histological chorioamnionitis/funisitis if delivery occurred within one week. Women with uterine contractions or evidence of another infection were excluded. Where possible, CRP results were extracted from the same day as the amniocentesis, or within 24 hours.