Objective
Placental growth factor and soluble Fms-like tyrosine kinase-1 may be potential diagnostic markers of preeclampsia. We compared performances of 2 immunoassays, the Triage placental growth factor assay and the Elecsys soluble Fms-like tyrosine kinase-1/placental growth factor ratio in diagnosing preeclampsia.
Study Design
A single site, case-control study of 44 patients with preeclampsia and 84 matched normal pregnant controls. Samples were collected at the time of diagnosis. Assays were performed according to product inserts.
Results
Both assays had optimal performance in diagnosing early-onset preeclampsia with area under the receiver operating characteristic curves of 0.99 (Triage: 100% sensitivity, 96% specificity; Elecsys: 64% sensitivity, 100% specificity for early-onset preeclampsia). Reassignment of the Elecsys cutoff for a positive test based on receiver operating characteristic curves increased sensitivity to 92%.
Conclusion
Using product insert cutoffs, Triage appears to have greater sensitivity at only a small reduction in specificity compared with Elecsys in the diagnosis of early-onset preeclampsia. A different cutoff may improve Elecsys sensitivity.
Preeclampsia is a leading cause of maternal and perinatal mortality and morbidity. Affecting 2-5% of pregnancies, it is usually characterized by hypertension (≥140/90 mm Hg) and proteinuria (≥300 mg/d) after 20 +0 weeks of gestation. However, preeclampsia is a heterogeneous syndrome and clinical presentation can be highly variable and may not include hypertension and proteinuria. The British Eclampsia Survey Team (BEST) found that only 57% of women with eclampsia had documented hypertension with proteinuria within the week before the first seizure. Women with nonproteinuric, preexisting, or gestational hypertension can evolve into preeclampsia and have been found to have worse outcomes than their classically defined preeclampsia counterparts. From the Preeclampsia Integrated Estimate of RiSk (PIERS) Study dataset, 254 of 2024 women with preeclampsia did not fulfill the research definition of preeclampsia on the day of their hospital admission. Likewise, of the 106 women who had an adverse maternal outcome within 48 hours of hospital admission, 21 (20%) had isolated HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and 6 (5%) had hyperuricemic hypertension (unpublished data). Mimickers of preeclampsia (eg, renal disease with preexisting hypertension and/or proteinuria) can also lead to clinical uncertainty. Women with nonclassically defined preeclampsia are still at risk for adverse outcomes and represent a clinically challenging subset of women to diagnose.
More comprehensive diagnostic tools are needed to aid in identifying women with atypical preeclampsia in clinical practice. In these situations, clinical symptoms may neither be sensitive nor specific to the diagnosis of preeclampsia. Biomarkers could provide an aid-to-diagnosis in women with suspected preeclampsia. Markers of placental origin, such as angiogenic factors, may be reasonable candidates for such diagnostic tests. Placental growth factor (PlGF) and its antagonistic receptor, Fms-like tyrosine kinase-1 (Flt-1) have recently emerged as 2 potential angiogenic markers for preeclampsia. Preeclampsia is associated with reduced maternal levels of PlGF and increased levels of soluble Flt-1 (sFlt-1). Changes in these factors also appear to reflect the severity of disease. Early-onset preeclampsia is associated with greater changes in PlGF and sFlt-1 compared with late-onset preeclampsia and normal pregnancy.
Recently, 2 automated immunoassays have become available for clinical use in the diagnosis of preeclampsia: the Triage PlGF assay (Alere, formerly Biosite, San Diego, CA) and the Elecsys sFlt/PlGF Ratio assay (Roche Diagnostics, Penzberg, Germany). We sought to compare their test performances in the diagnosis of preeclampsia to determine which could be the most suitable for application in routine clinical practice.
Materials and Methods
Study population
In this retrospective, case-control study, blood samples were collected prospectively from women following written, informed consent, between November 2004 and August 2007 at BC Women’s Hospital in Vancouver, Canada. Ethics approval was granted by the University of British Columbia Children’s and Women’s Health Centre Research Ethics Boards. Other data pertaining to this cohort of women have previously been described. Women with preeclampsia were recruited consecutively from inpatient and outpatient services at BC Women’s Hospital (eg, Delivery Suite, Assessment, Ultrasound, Family Practice Maternity Services, Maternal Fetal Medicine Clinic). Normal pregnancy controls were matched to cases during the recruitment period based on maternal age (± 5 years), gestational age (± 2 weeks), and parity (0, ≥1). Women (both cases and controls) were excluded if they were in active labor at the time of eligibility or had received antenatal betamethasone within 48 hours.
Preeclampsia was defined as hypertension (blood pressure ≥140/90, on at least 2 occasions >4 hours apart after 20 weeks’ gestation) and new onset proteinuria (≥2+ dipstick reading, ≥0.3 g/d by 24-hour urine collection, or ≥30 mg/mmol by protein:creatinine ratio). Blood pressure measurements were taken using a mercury sphygmanometer, with Korotkoff V used to determine diastolic pressure. Women were positioned in a semirecumbent position, with a supported arm and appropriately sized cuff. Small-for-gestational-age status of the fetus was determined if birthweight was <5th percentile for gestational age and sex. Normal pregnancy was defined as term delivery with no documented concerns of hypertension, proteinuria, gestational diabetes, or growth restriction during the current pregnancy and was confirmed after delivery. For both cases and controls, blood pressure measurements were taken at the time of blood sample collection.
A total of 128 women were included in this study: 44 women with preeclampsia and 84 normal pregnancy controls. Of the preeclampsia cases, 25 women had early-onset preeclampsia defined as preeclampsia diagnosed ≤34 weeks’ gestation. Nineteen women had late-onset preeclampsia defined as preeclampsia diagnosed >34 weeks’ gestation. This classification is consistent with the American Society of Hypertension’s onset criteria of early and late preeclampsia. Of the controls, 47 had blood collected ≤34 weeks’ gestation (early-onset controls) and 37 had blood collected >34 weeks’ gestation (late-onset controls).
Clinical specimens and angiogenic factor analysis
Venous blood was collected at the time a confirmed diagnosis of preeclampsia was made. Serum was collected using silicon-coated glass tubes. Plasma was collected in EDTA tubes. Samples were prepared by centrifugation and frozen at −80°C. Laboratory staff was blinded to clinical diagnosis. Samples were batch assayed to minimize any effect of interassay variability.
Plasma was analyzed for PlGF using the Triage PlGF assay according to the manufacturer’s instructions. Using fluorescently labeled monoclonal antibodies against PlGF for PlGF quantification, this immunoassay uses a single use disposable plastic assay test cartridge in conjunction with the Triage Meter (Biosite Incorporated, San Diego, CA). Briefly, 250 μL thawed plasma (room temperature) is pipetted into the sample port of a new test cartridge. The cartridge is inserted into the meter and results are displayed in approximately 15 minutes in pg/mL. The cartridge contains chemistries for onboard positive and negative control systems. Control systems at the level of the cartridge and meter ensure that the quantitative PlGF result is valid. Calibration information is supplied by the manufacturer in the form of a lot-specific EPROM chip that is contained within each kit of devices. The measurable range of the assay is 12-3000 pg/mL. Concentrations below 12 pg/mL are value assigned based on the calibration curve, but this value is displayed to the user as a qualitative result “<12 pg/mL.” A positive test was defined as a PlGF concentration <5th percentile for gestational age of normal controls, as described in the product insert.
Serum was analyzed for PlGF and sFlt-1 using the Elecsys assay to obtain an sFlt-1/PlGF ratio, according to the manufacturer’s instructions. Detection limits for sFlt-1 range from 10–85,000 pg/mL and 3–10,000 pg/mL for PlGF. A fixed (independent of gestational age) ratio >85 was used as the cutoff for a positive test as described in the product inset. In addition, gestational age dependent sFlt-1/PlGF ratios from normal pregnancy were used to derive gestational age dependent cutoffs for a positive test (above the 95th percentile for gestational age range from normal pregnancy).
Statistics
Data were analyzed using Prism 4.0 (GraphPad, San Diego, CA) and SPSS 18.0 (SPSS Inc, Chicago, IL). Descriptive data were expressed as medians with interquartile ranges (IQR) for nonnormally distributed data. The χ 2 tests were used for comparison of categoric variables. Mann-Whitney tests were used for continuous variables.
Receiver operator characteristic (ROC) curves were constructed to calculate area under the curves (AUC) and used to evaluate the performance of each assay in the diagnosis of preeclampsia at any gestational age, early gestational age (≤34 weeks), and late gestational age (>34 weeks). The ROC curve for diagnosis at any gestational age was used to determine a fixed cutoff for each assay which maximized sensitivity and specificity. Sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) were calculated using product insert cutoffs, fixed cutoffs derived from the ROC curves, and gestational-age dependent ratio cutoffs (Elecsys only). P values < .05 were judged to be statistically significant. PPV and NPV were calculated to characterize the 2 × 2 tables, despite the artificial prevalence in this case-control study. Also, qualitative PlGF results below 12 pg/mL were set equal to 12 pg/mL for the purpose of statistical analysis. This approximation does not affect the reported test performance in terms of clinical sensitivity, or specificity, but may slightly under report the significance of the difference between groups in the Mann-Whitney analysis, or in the ROC AUC analysis.
Comparison of these diagnostic tests followed the STARD (Standards for Reporting of Diagnostic Accuracy) Initiative guidelines.
Results
Clinical characteristics
Clinical characteristics of women with preeclampsia and their matched controls are shown in Table 1 . Matching criteria (maternal age, parity, gestational age) did not differ between the groups. Higher blood pressure, more proteinuria, and a trend toward lower platelets were seen in cases compared with controls. Gestational age at delivery was lower, preterm birth, and small-for-gestational age fetuses more common in cases compared with controls.
| Characteristic | Early-onset PET (n = 25) | Early-onset Ctrl (n = 47) | Late-onset PET (n = 19) | Late-onset Ctrl (n = 37) | P value (χ 2 or KW) |
|---|---|---|---|---|---|
| Baseline | |||||
| Maternal age, y | 36 [31–40] | 34 [32–35] | 33 [31–37] | 33 [30–36] | .3 |
| Nulliparous | 15 (60) | 25 (53) | 16 (84) | 31 (84) | .007 |
| GA at sampling, wks | 32.3 [28.5–33.3] | 31.9 [27.1–33.7] | 37.1 [35.6–38.4] | 36.9 [36.1–38.3] | < .0001 |
| Multiple pregnancy | 3 (12) | 1 (2) | 1 (5) | 0 (0) | .1 |
| sBP at sampling, mm Hg | 160 [157–182] | 116 a [108–125] | 160 [150–175] | 118 b [110–122] | < .0001 |
| dBP at sampling, mm Hg | 107 [100–115] | 76 a [70–85] | 100 [100–110] | 70 b [70–84] | < .0001 |
| Proteinuria | 25 (100) | 0 (0) | 19 (100) | 0 (0) | < .0001 |
| Platelets (×10 9 /L) | 186 [168–227] | 221 (n = 23) [172–258] | 158 [124–216] | 214 (n = 20) c [185–257] | .005 |
| Uric acid, μM | 347 [296–410] | — | 380 [335–410] | — | .30 d |
| AST, μM | 32 [28–44] | — | 33 [26–57] | — | .91 d |
| Outcome | |||||
| GA at delivery | 32.9 [31.6–34.4] | 39.7 a [38.1–40.3] | 37.4 [36.1–39.0] | 39.7 e [39.0–40.3] | < .0001 |
| Preterm birth (<37 +0 wks) | 24 (86) | 0 (0) | 5 (25) | 0 (0) | < .0001 |
| Birthweight, g (n = fetuses) | 1650 (n = 28) [1400–1785] | 3496 a (n = 48) [3185–3790] | 2730 (n = 20) [2225–3310] | 3428 c (n = 37) [3055–3710] | < .0001 |
| SGA (<5th percentile) | 6 (21) | 0 (0) | 6 (30) | 0 (0) | < .0001 |
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