Objective
The angiogenic and antiangiogenic factors soluble fms-like tyrosine kinase (sFlt)-1 and placental growth factor (PIGF) have been implicated in the mechanisms of disease responsible for preeclampsia (PE). Moreover, it has been proposed that the concentrations of these markers in maternal serum/plasma may have predictive value. This study evaluates a newly developed Elecsys (Roche, Penzberg, Germany) assay for sFlt-1 and PIGF and tests the value of the sFlt-1/PIGF ratio in the assessment of PE.
Study Design
This multicenter case-control study included 351 patients: 71 patients with PE and 280 gestational age-matched control subjects from 5 European study centers. A total of 595 serum samples were measured for sFlt-1 and PIGF using an automated platform.
Results
Maternal serum concentrations of sFlt-1 and PIGF significantly separated healthy women and women with PE. The sFlt-1/PIGF ratio had an area under the receiver operating characteristic curve of 0.95. The best performance was obtained in the identification of early-onset PE (area under the receiver operating characteristic curve of 0.97).
Conclusion
Measurement of sFlt-1 and PIGF and calculation of sFlt-1/PIGF ratio can be performed quickly and in a platform available in clinical laboratories. This is a substantial step forward in bringing the determination of these analytes to clinical practice in obstetrics. We propose that sFlt-1, PIGF, and sFlt-1/PIGF ratio may be of value in the prediction of PE and in the differential diagnosis of patients with atypical presentations of PE, and perhaps in the differential diagnosis of women with chronic hypertension suspected to develop superimposed PE.
Preeclampsia (PE) is still a leading cause of fetal and maternal morbidity and mortality with an incidence of 3-5% worldwide. PE accounts for 42% of all maternal deaths per year and is associated with 15% of all preterm deliveries. Despite intensive research efforts the pathogenesis of the disease is still unknown but it is likely to be multifactorial.
The diagnosis of PE is based on the measurement of blood pressure and proteinuria. However, sensitivity and specificity of these definitions are low regarding the prediction of adverse maternal and fetal outcomes. The clinical presentation of the disease is variable, comprising severe and rapidly progressing early-onset PE with the need to end pregnancy and deliver a preterm baby or mild forms of late-onset PE at term.
The reliable identification of high-risk PE patients is crucial as intensified monitoring and referral to specialized perinatal care centers substantially reduces maternal and fetal morbidity. Quick and reliable detection of the disease allows expeditious intervention with steroids for fetal lung maturity, magnesium for seizure prophylaxis, antihypertensive therapy, and bed rest.
Changes in the serum concentrations of angiogenic and antiangiogenic factors are implied in the pathogenesis of PE and have possible relevance in the diagnosis of the disease. Elevated serum concentrations of the antiangiogenic soluble fms-like tyrosine kinase (sFlt)-1 receptors are involved in PE. However, serum concentrations of the angiogenic placental growth factor (PIGF) are reported to be decreased in women with PE. It was observed that sFlt-1 concentrations increased beginning approximately 5 weeks before the onset of PE and PIGF expression declined already at 13-16 weeks of gestation suggesting possible use as a screening parameter. For this reason various investigators have examined the use of these factors as possible predictors for PE.
With accumulating evidence for sFlt-1 and PIGF as an aid in diagnosis as well as an aid in prediction for PE, it is necessary to have a quick and reliable test for these parameters that is feasible in the clinical setting. While enzyme-linked immunoassay (ELISA) kits for the measurement of sFlt-1 and PIGF for experimental use are available, no such means for the clinical context and widespread use was evaluated up to now. The objective of the study was to describe the behavior of sFlt-1 and PIGF using the Elecsys (Roche, Penzberg, Germany) platform in PE and normal pregnancy. We wished to provide reference ranges for the use of the sFlt-1/PIGF ratio and to determine whether use of this ratio was superior to use of the individual biomarkers sFlt-1 and PIGF in the assessment of PE.
Materials and Methods
Study population
Singleton pregnancies were enrolled at 5 participating European medical centers. An identical study protocol and data collection form was used at each center. The local ethics committees and institutional review boards approved the procedure, and all subjects gave their written, informed consent before participation. Background data on the patients were provided by the study centers in form of a completed case report form. A total of 351 individuals were enrolled in the study: 280 singleton pregnancies with normal pregnancy outcome and 71 singleton pregnancies with PE outcome.
This study consists of 2 major arms. The first study arm is a prospective cohort study of healthy pregnant women to determine the reference range for sFlt-1, PIGF, and the sFlt-1/PIGF ratio. All 280 women with healthy singleton pregnancies were enrolled in this study. The women were asked to donate samples of blood at the time of admission to the labor and delivery unit or at routine visits in the outpatient unit. For these patients follow-up visits (further on termed “visits”) were conducted whenever possible and additional blood was collected. Thus, serum specimens were collected in 524 visits from control patients (mean visit/patient 1.87, visits/patient differ from 1-7). For the establishment of gestational age-dependent reference ranges, gestational age intervals were formed as follows: interval 1, gestational weeks 10-14; interval 2, weeks 15-19; interval 3, weeks 20-23; interval 4, weeks 24-28; interval 5, weeks 29-33; interval 6, weeks 34-36; and interval 7, weeks 37-delivery. The number of blood samples used to determine the gestational age interval-specific reference range differs from a minimum of 40 to a maximum of 105 visits. Reference ranges for the Elecsys assays and the sFlt-1/PIGF ratio were specifically developed for these gestational time periods. Maximal 1 visit per patient per gestational age interval was collected in case of follow-up.
Inclusion criteria for the prospective cohort study arm were the presence of informed consent, maternal age >16 years, and a normal pregnancy outcome. A normal pregnancy outcome refers to the mother not diagnosed with any form of PE and the infant not diagnosed with intrauterine growth retardation (IUGR). The exclusion criteria comprise the diagnoses of PE, HELLP syndrome, IUGR, and all those for the PE group.
The second arm of this study is a case-control study to determine the relationship between serum concentrations of sFlt-1 and PIGF in women diagnosed with PE and those with normal pregnancies. All 71 singleton pregnancies with PE outcome and 268 control patients were enrolled in the study (12 women of the 280 with normal pregnancy outcome were enrolled only for the reference range study but not for the case-control study as they contributed samples exclusively before gestational week 20). The patients of the control group contributed 462 visits (starting from gestational week 20) for the case-control comparison and the receiver operating characteristic (ROC) analysis. In the PE group, only the first visit when PE was diagnosed was included resulting in a total of 71 visits. The PE group was further divided into early-onset and late-onset PE in accordance to the onset of the disease before or after 34 + 0 weeks of gestation. The controls were matched according to gestational age and maternal characteristics.
Inclusion criteria for the PE group were the presence of informed consent, maternal age >16 years, and the presence of PE according to the definitions stated below. Exclusion criteria were loss to follow-up, multigestation, antiphospholipid antibody syndrome, systemic lupus erythematosus, or any other autoimmune disease as well as chronic corticosteroid or nonsteroidal antiinflammatory drug use except low-dosage aspirin <150 mg/day.
PE was defined according to the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Under this classification, PE is defined as blood pressure ≥140 mm Hg systolic or ≥90 mm Hg diastolic in a woman who was normotensive <20 weeks’ gestation on at least 2 measurements within a 3-day period. Significant proteinuria is defined as ≥0.3 g protein/24 h (or in emergency cases only a dipstick ≥1+ on >1 occasion or the determination of the ratio of sFlt1/PIGF can be performed using the Elecsys platform, and results are informative of the balance of selective antiangiogenic and angiogenic factors in PE; 30 mg/dL protein in a spot urine if a 24-hour urine protein collection can not be obtained).
All patients who fulfilled the defined diagnostic criteria for PE were enrolled, regardless of further subclassifications. HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) was diagnosed when increased transaminase (aspirate aminotransferase, alanine aminotransferase concentrations >2 × upper reference interval), reduced thrombocyte counts (<100,000/μL) plus at least hemolysis criterion (increased lactate dehydrogenase concentrations >2 × upper reference interval) or indirect bilirubin serum concentrations >1.2 mg/dL or reduced haptoglobin serum concentrations <0.3 g/L) were measured. Superimposed PE was diagnosed in a patient with preexisting hypertension with de novo proteinuria >20 weeks’ gestation or sudden increase in the magnitude of hypertension or the appearance of thrombocytopenia and/or abnormal transaminases or in preexisting proteinuria, a sudden increase in proteinuria. Patients with preexisting chronic hypertension (patient previously diagnosed with hypertension and currently receiving antihypertensive medication; patient who has never been diagnosed with hypertension, but develops hypertension <20 weeks’ gestation; and patient with hypertension that persists >12 weeks’ postpartum) as well as preexisting proteinuria were also enrolled in the PE group. For analysis, no distinction was made among PE, severe PE, HELLP syndrome, or superimposed hypertension/proteinuria as patient numbers were too low to sufficiently form subgroups. IUGR in the applied study protocol was defined as estimated fetal weight or abdominal circumference below the fifth percentile (adjusted for gender and ethnicity according to the charts routinely used by the study site) in the presence of oligohydramnion (amniotic fluid index <10th percentile) or pathologic flow in umbilical artery (pulsatility index >95th percentile).
Samples
Serum samples were collected according to a common standard operating procedure at each center. Maternal blood was collected by venipuncture in tubes without anticoagulant. Serum was allowed to form, and then the samples were centrifuged with 2000 g , pipetted, and stored at –80°C until testing. The sFlt-1 and PIGF concentrations of each sample were determined in parallel. Complete testing was performed with an Elecsys 2010 analyzer. For each sample the sFlt-1/PIGF ratio was calculated. Histograms and basic statistics (mean, median, SD, quantiles, and range) are given for markers sFlt-1, PIGF, and sFlt-1/PIGF ratio.
Immunoassays
Single measurements were performed for sFlt-1 and PIGF on the fully automated Elecsys system (Elecsys PIGF, human PIGF and Elecsys sFlt-1, sFlt-1). Both assays are sandwich immunoassays based on the electrochemiluminescence technology. The total duration of the assays is 18 minutes and the sample volume is 20 μL for sFlt-1 and 50 μL for PIGF. The assays are calibrated with recombinant human PIGF and sFlt-1 and standardized against the Quantikine PIGF and vascular endothelial growth factor-R1 ELISAs (R&D Systems, Minneapolis, MN). According to the manufacturer’s instructions for use, the Elecsys sFlt-1 assay covers a measuring range from 10-85,000 pg/mL, the Elecsys PIGF assay from 3-10,000 pg/mL. The limit of detection is 10 pg/mL (sFlt-1) and 3 pg/mL (PIGF). The limit of quantitation (functional sensitivity) is <15 pg/mL for sFlt-1 and 10 pg/mL for PIGF. Within-run imprecision coefficients of variation (CV) determined for the control samples of the assays are <2% for sFlt-1 and PIGF. Between-run CVs range from 2.6–3.0% for sFlt-1 and from 2.0–2.4% for PIGF.
Statistical analysis
To compare clinical groups, scatterplots were generated for the serum concentrations of sFlt-1, PIGF, and sFlt-1/PIGF ratio. Gestational age-dependent reference values were calculated per time interval as quantiles. In addition, smooth curves for gestational age-dependent quantiles were determined using a robust quantile regression method. Absolute values are given in mean ± SE. Statistical comparisons of the serum concentrations of different analytes in cases and controls were performed using parametric or nonparametric methods. A P value of < .05 was considered significant. All P values reported are 2-tailed.
For the prospective cohort study arm the changes in maternal serum concentration as a function of gestational age was examined (gestational age range, 10–43 weeks). If a given patient had >1 visit, the first visit was the one used for the construction of the reference range.
For the case-control study arm, ROC curve was used for the evaluation of the area under the curve (AUC) as well as the sensitivity and false-positive rate for different cutoffs of the analytes sFlt-1, PIGF, and the sFlt-1/PIGF ratio, respectively. The different AUCs were compared statistically.
Results
Demographic and clinical characteristics of the population
For the reference range study arm a total of 280 patients were included. For the case-control study arm 71 women with clinical PE and 268 women with uneventful pregnancies were included. No significant differences in age, gestational age at enrollment, or ethnical origin were observed. Women with PE had a higher systolic and diastolic blood pressure, had a lower mean birthweight of the neonate, and were less likely to smoke than normotensive women. The results are listed in Table 1 .
| Characteristic | Group | ||
|---|---|---|---|
| Early onset PE | Late onset PE | Control | |
| Age, y | 31.1 ± 5.2 | 29.4 ± 6.9 | 30.6 ± 5.8 |
| Height, cm | 165.5 ± 7.6 | 165.6 ± 7 | 166.2 ± 6.7 |
| Weight, Kg | 71.3 ± 24.5 | 69.7 ± 17 | 66.5 ± 15.5 |
| BMI, kg/m 2 | 26.1 ± 7.8 | 25.4 ± 6.1 | 24.1 ± 5.5 |
| Birthweight, g | 1388 ± 707.2 a | 2710.6 ± 679.2 a | 2977.3 ± 810.9 |
| Gestational week of delivery | 30.7 ± 3.3 | 37.7 ± 2.1 | 37.2 ± 3.6 |
| Diastolic BP, max mmHg b | 99 ± 13.2 a | 97.9 ± 11 a | 74.6 ± 11.3 |
| Systolic BP, max mmHg b | 153.9 ± 16.2 a | 152.6 ± 18.1 a | 118.8 ± 15.7 |
| Smoking status, n (%) | |||
| Current | 3 (8.1) | 3 (8.8) | 46 (17.2) |
| Past | 1 (2.7) | 3 (8.8) | 58 (21.6) |
| Never | 31 (83.8) | 22 (64.7) | 127 (47.4) |
| Unknown | 2 (5.4) | 6 (17.6) | 37 (13.8) |
| Race or ethnic group, n (%) | |||
| White/Caucasian | 32 (86.5) | 28 (82.4) | 242 (90.3) |
| Black/African American | 1 (2.7) | 1 (2.9) | 5 (1.9) |
| Other | 4 (10.8) | 3 (8.8) | 17 (6.3) |
| Unknown | 0 (0) | 2 (5.9) | 4 (1.5) |
| Family history of PE, n (%) | |||
| Yes | 2 (5.4) | 3 (8.8) | 3 (1.1) |
| No | 28 (75.7) | 22 (64.7) | 233 (86.9) |
| Unknown | 7 (18.9) | 9 (26.5) | 32 (11.9) |
| Total N | 37 | 34 | 268 |
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