Subjects and specimens

Pregnant women were enrolled in this prospective study at 2 clinical sites: 1 in Minneapolis, MN, and 1 in Toledo, OH. Enrollment began in December of 2006 and ended in January of 2008. The protocol was approved by each organization’s institutional review board, and all subjects provided written informed consent. Subjects were eligible for enrollment if they were between 16 and 45 years of age. The 2 arms of the study included: (1) women presenting with signs and symptoms of preeclampsia between gestational ages 20-36 weeks (cases), and (2) normal apparently healthy subjects as well as those with increased risk for preeclampsia (controls). For the latter arm, subjects were to be enrolled with their first visit occurring at 16-20 weeks of gestation, a second visit occurring in conjunction with their clinic visit for glucose tolerance testing, and a third (final) visit corresponding to admission for delivery. Blood specimens, as well as demographic and clinical data, were collected at each visit. Blood pressures were taken and documented at each study visit and other clinical assessments were performed as deemed appropriate by the attending physician. Other clinical assessments may have included urine protein determinations (by dipstick or 24-hour collection), platelet counts, hemoglobin, serum creatinine, serum uric acid, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Prescription and over-the-counter medications and dietary supplements were also documented. EDTA plasma was separated and stored at -70° C until it was tested for sVEGF R1 and PlGF. Pregnancy outcomes, including maternal and neonatal health information, were documented.

Preeclampsia was diagnosed by the attending physician during or shortly after the visit in which the subject presented with signs and symptoms and was in accordance with the American College of Obstetrics and Gynecology practice bulletin. Briefly, hypertension was defined as a systolic blood pressure >140 mmHg or a diastolic >90 mmHg, and proteinuria was defined as urinary excretion of 0.3 g protein or greater in a 24-hour urine specimen or when urinary dipstick reading was 2+ or greater. Subjects with chronic hypertension were diagnosed with superimposed preeclampsia if proteinuria reached or exceeded 0.5 g protein in a 24-hour urine specimen or a dipstick reading of 2+ or greater or if the subject had thrombocytopenia (platelet count <150,000/μL) or elevated liver enzymes (AST >70 IU/L). Thirty subjects diagnosed with preeclampsia had 24-hour urine specimens while the remaining 18 subjects were evaluated with dipstick proteinuria.

Biomarker assays

EDTA plasma specimens were analyzed for free sVEGF R1 and free PlGF using automated prototype assays for the Beckman Coulter family of Access immunoassay systems. These prototype assays are under development and not available for clinical use. The Access PlGF and sVEGF R1 assays are one-step immunoenzymatic (“sandwich”) assays. A sample is added to a reaction vessel along with paramagnetic particles coated with mouse monoclonal antihuman PlGF or mouse monoclonal antihuman sVEGF R1, blocking agent and a second monoclonal antibody conjugated to alkaline phosphatase. After incubation, materials bound to the solid phase are held in a magnetic field while unbound materials are washed away. The chemiluminescent substrate Lumi-Phos 530 is added and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of PlGF or sVEGF R1 in the sample, with the amount of analyte in the sample determined from a stored, multi-point calibration curve. Reagents for both Access assays were provided in ready-to-use reagent cartridges and the assays were run on Access 2 automated immunoassay analyzers at the clinical sites. The upper limit of the measuring range for the prototype PlGF assay was 10,000 pg/mL while the upper limit of the measuring range for the prototype sVEGF R1 assay was 132,500 pg/mL. Within run and between run precision has been determined previously with <3% CV for each assay.

Statistical analyses

Diagnostic cutoff values were determined by receiver operating characteristics (ROC) analysis and clinical sensitivities and clinical specificities were calculated using 2 × 2 contingency analyses. Median sVEGF R1 and PlGF concentrations were determined and nonparametric Mann-Whitney U tests were used to test for differences between experimental groups. Student t test and Fisher exact test were used in the analyses of the maternal and infant characteristics reported in Table 2 . Differences with P values of < .05 were considered statistically significant.