Preeclampsia

New Gestational PhaseSpecific Cutoff Values for the Use

of the Soluble fms-Like Tyrosine Kinase-1/Placental Growth
Factor Ratio as a Diagnostic Test for Preeclampsia
Stefan Verlohren, Ignacio Herraiz, Olav Lapaire, Dietmar Schlembach, Harald Zeisler,
Pavel Calda, Joan Sabria, Filiz Markfeld-Erol, Alberto Galindo, Katharina Schoofs,
Barbara Denk, Holger Stepan
See Editorial Commentary, pp 210211
AbstractTo establish gestational phase adapted cutoffs for the use of the soluble fms-like tyrosine kinase-1 (sFlt-1)/placental
growth factor (PlGF) ratio as a diagnostic tool for preeclampsia in the clinical setting, a multicenter casecontrol study
including a total of 1149 patients was performed. We report normal values of sFlt-1, PlGF, and the sFlt-1/PlGF ratio based
on the analysis of a total of 877 patients with uneventful pregnancy outcome. A total of 234 patients with preeclampsia and
a matched cohort consisting of 468 patients with normal pregnancy outcome were compared, and sFlt-1 and PlGF were
measured on an automated platform. Separate cutoffs for the sFlt-1/PlGF ratio were determined for the early (20+033+6
weeks) and the late gestational phase (34+0 weeksdelivery). For each of the 2 gestational phases, 2 independent cutoffs
framing an equivocal zone were determined: the first cutoff with focus on high sensitivity, and the second focusing on high
specificity. Between 20+0 and 33+6 weeks, the cutoffs at 33 and 85 resulted in a sensitivity/specificity of 95%/94% and
88%/99.5%, respectively. An sFlt-1/PlGF ratio of 33 had the lowest likelihood of a negative test (0.05; 95% confidence
interval, 0.020.13), whereas values 85 had the highest likelihood of a positive test (176; 95% confidence interval, 24.88
1245). After 34+0 weeks, the cutoffs at 33 and 110 yielded a sensitivity/specificity of 89.6%/73.1% and 58.2%/95.5%,
respectively. The approach to use multiple cutoffs for the early and late gestational phase enhances the diagnostic accuracy of
the sFlt-1/PlGF ratio as a diagnostic tool for preeclampsia.(Hypertension. 2014;63:346-352.) Online Data Supplement

Key Words: angiogenesis angiogenic factors antiangiogenic factors hypertension preeclampsia

pregnancy sFlt1-PlGF ratio

reeclampsia is a multisystem disorder in pregnancy. With

an incidence of 2% to 5% of all pregnancies, it is a frequent
pregnancy-related disease and a major cause of maternal and fetal
morbidity and mortality.1 Preeclampsia is defined as the newonset of hypertension (140/90 mmHg on 2 separate occasions
4 hours apart) and proteinuria (300 mg/24 h).2 The simplicity
of this definition contrasts with the complexity of the disease. The
pathophysiology of preeclampsia is not conclusively resolved up
to now. Early-onset preeclampsia, defined as the onset before 34
weeks of gestation, comprises the malimplantation of the placenta, insufficient spiral-artery remodeling, prevalently resulting in intrauterine growth restriction and an altered expression
of placental proteins such as soluble fms-like tyrosine kinase-1

(sFlt-1) and placental growth factors (PlGFs).3 In contrast, lateonset preeclampsia, defined as the onset after 34 weeks of gestation, is not necessarily accompanied by placental dysfunction4
but angiogenic and antiangiogenic factors are also dysregulated,
though to a less dramatic extent.5
We and others have previously shown that the sFlt-1/PlGF
ratio is elevated in patients with preeclampsia.610 The automated measurement of the sFlt-1/PlGF ratio detects early-onset
preeclampsia with a sensitivity of 89% and a specificity of 97%
when the single, gestation-wide cutoff of 85 is used.8 Recently,
Rana et al9 have shown that in patients with signs and symptoms
for the disease, an sFlt-1/PlGF ratio 85 predicts the occurrence
of preeclampsia-related adverse maternal and fetal outcomes,

Received June 15, 2013; first decision July 7, 2013; revision accepted October 1, 2013.
From the Department of Obstetrics, Campus Virchow-Clinic, Charit University Medicine Berlin, Berlin, Germany (S.V., K.S.); Fetal Medicine Unit,
Department of Obstetrics and Gynecology, Hospital Universitario 12 de Octubre, Universidad Complutense de Madrid, Madrid, Spain (I.H., A.G.);
Department of Obstetrics and Gynecology, University of Basel, Basel, Switzerland (O.L.); Department of Obstetrics and Gynecology, University of Jena,
Jena, Germany (D.S.); Department of Obstetrics and Gynecology, University of Vienna Medical School, Vienna, Austria (H.Z.); Department of Obstetrics
and Gynecology, Charles University, Prague, Czech Republic (P.C.); Department of Obstetrics and Gynecology, University Hospital Sant Joan de Du,
Espluges, Spain (J.S.); Department of Obstetrics and Gynecology, University of Freiburg, Freiburg, Germany (F.M.-E.); Roche Diagnostics, Clinical
Operations Professional Diagnostics, Penzberg, Germany (B.D.); and Department of Obstetrics, University Hospital Leipzig, Leipzig, Germany (H.S.).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
113.01787/-/DC1.
Correspondence to Stefan Verlohren, Department of Obstetrics, Charit University Medicine Berlin, Charitplatz 1, D-10117 Berlin, Germany. E-mail
stefan.verlohren@charite.de
2013 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org

DOI: 10.1161/HYPERTENSIONAHA.113.01787

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by guest on August 26, 2015
346

Verlohren et al Preeclampsia and sFlt-1/PlGF Ratio 347

irrespective of the presence of a diagnosis of preeclampsia
according to the gold standard.
With accumulating evidence of the importance of the sFlt-1/
PlGF ratio as a diagnostic and prognostic marker, the limitations
and benefits of its clinical use have to be assessed. Up to now,
only 1 cutoff (85) exists, irrespective of the gestational week
tested, with limited diagnostic accuracy especially in late-onset
preeclampsia. This cutoff was based on a preliminary analysis of
a multicenter casecontrol study consisting of 71 patients with
preeclampsia and a matched cohort of 280 control patients.
Here, we report the final analysis of a multicenter casecontrol study including a total of 1149 patients. We report normal
values of sFlt-1, PlGF, and the sFlt-1/PlGF ratio based on the
analysis of a total of 877 patients with uneventful pregnancy
outcome. To better reflect the putatively different pathophysiology of early-onset and late-onset preeclampsia, diagnostic
accuracy of 2 separate cutoffs framing an equivocal zone was
determined for the early and late phase of gestation.

Methods
Study Population
Singleton pregnancies were enrolled at 9 European perinatal care
centers. An identical study protocol and data collection form was
used at each center. The local ethics committees and institutional
review boards approved the study, and all subjects gave their written
informed consent before participation.
Preeclampsia was defined according to the National High Blood
Pressure Education Program Working Group on High Blood Pressure in
Pregnancy.11 The syndrome of hemolysis, elevated liver enzymes, and
low platelets (HELLP syndrome), superimposed preeclampsia, and intrauterine growth restriction were defined as previously published.8 In
this study, patients with any form of preeclampsia or HELLP were combined in the preeclampsia/HELLP group (preeclampsia/HELLP) for
analysis. A pregnancy outcome was defined as normal if the mother was
not diagnosed with any form of preeclampsia/HELLP and the infant
was not diagnosed with intrauterine growth restriction. Investigators
were blinded to sFlt-1 and PlGF levels which prevented an influence of
this information on decision making and defining time point of delivery.
A total of 1149 individuals were enrolled in the study: 915 singleton
pregnancies with normal pregnancy outcome and 234 singleton pregnancies with preeclampsia outcome. At least 100 preeclampsia/HELLP
cases for each early and late gestational phase were determined by
study protocol as target numbers for the nested casecontrol population. Recruitment was performed in a prospective manner until these
targets were obtained with the consequence of enrolling 1149 patients.
Two subsets of this data collective were built (Figure S1 in the onlineonly Data Supplement). For analysis of reference ranges, maximum 1
visit from each subject per gestational age window was included in case
of follow-up visits in the control group. Therefore, this subset contains
1685 control visits from 877 subjects. An additional 38 control subjects
either contributed samples only before week 10 or were not normotensive and were therefore excluded from the reference range analysis. For
the determination of gestational agedependent reference values, we
analyzed 7 gestational age windows: window 1: 10+0 to 14+6 weeks
of gestation; window 2: 15+0 to 19+6 weeks of gestation; window 3:
20+0 to 23+6 weeks of gestation; window 4: 24+0 to 28+6 weeks of
gestation; window 5: 29+0 to 33+6 weeks of gestation; window 6: 34+0
to 36+6 weeks of gestation; and window 7: 37+0 weeks of gestation to
delivery. All gestational age windows comprise 5 weeks except window
3 (2023+6 weeks of gestation) and window 6 (34+036+6 weeks of
gestation), acknowledging clinical thresholds at 24+0 (viability), 34+0
(early/late preeclampsia), and 37+0 weeks of gestation (preterm birth).
For receiver operating characteristics (ROC) curves and analysis of the
cutoff, a total of 234 subjects with a preeclampsia/HELLP outcome were
matched to 468 subjects with uneventful pregnancy outcomes. Only visits
from subjects with a gestational age of 20+0 weeks were included. In
patients with preeclampsia/HELLP, only the first visit after confirmation

of the diagnosis was included in the analysis. A preeclampsia pregnancy

was defined as early onset if preeclampsia was diagnosed at a visit before
week 34 (33+6 weeks of gestation). In case preeclampsia was diagnosed
at >34+0 weeks of gestation, the patient was assigned to the late-onset
preeclampsia group. Patients of the preeclampsia/HELLP group were
matched pairwise by gestational week to a healthy control in a 1:2 manner,
resulting in a double sample size of the control group. A control patient
only donated samples either to the early- or the late-onset preeclampsia
group, and no repeated samples were used in the casecontrol cohort.
In a separate descriptive subgroup analysis, patients with superimposed preeclampsia (n=14), isolated HELLP (n=15), preeclampsia
not being severe or complicated by HELLP syndrome (n=99), and
patients with severe preeclampsia (n=106) were analyzed.

Samples and Immunoassays

Serum samples were collected according to a common standard operating procedure at each center. Single measurements were performed for
sFlt-1 and PlGF on the fully automated Roche Elecsys system (Elecsys
PlGF, human PlGF, and Elecsys sFlt-1, sFlt-1) as described previously,
and the sFlt-1/PlGF ratio was calculated for each sample.12,13 Only serum
samples were analyzed in this study, results in plasma samples may differ.

Statistics
Basic statistics (mean, median, SD, quartiles, and range) were performed
for sFlt-1, PlGF, and the sFlt-1/PlGF ratio. To compare clinical subgroups, descriptive statistics (medianinterquartil ranges) were generated. Gestational agedependent reference values were determined per
time window as quantiles. For the statistical comparison of marker levels
in the respective clinical groups, data were transformed as appropriate
and either parametric (ANOVA, t test) or nonparametric (Wilcoxon/
KruskalWallis) tests were applied. All P values are 2-tailed. Statistical
significance was assigned when a P value was <0.05. All statistical analyses were performed using SAS. ROC analysis was used for the evaluation
of the area under curve and the sensitivity and specificity as a function of
cutoff for markers sFlt-1, PlGF, and sFlt-1/PlGF ratio. Positive and negative likelihood ratios (LR+/LR) were calculated. Gerhard plots were
generated to find optimized cutoff values for the single markers and the
ratio. A generalized linear regression model with a -distributed response
regarding the positively skewed, continuous distribution was applied to
test for confounding effects of body mass index and maternal age.

Results
Demographic and Clinical Baseline Characteristics
No significant differences were found in age, height, and
weight between patients with early- or late-onset preeclampsia and their gestational agematched controls. As expected,
patients with preeclampsia had a significantly earlier date of
delivery, higher systolic and diastolic blood pressure, higher
body mass index, and lower neonatal birth weight (P<0.05 or
P<0.001 where appropriate, Table S1).

Gestational AgeDependent Normal Values

Normal values for the sFlt-1/PlGF ratio as well as the single
markers were generated in 7 gestational age windows from
women with uneventful pregnancy outcome (Table1). As ex
pected, the sFlt-1/PlGF ratio showed an U-shaped curve with
median sFlt-1 concentrations rising and PlGF values exhibiting a
bell-shaped curve in the course of pregnancy (Figure S2aS2c).

sFlt-1 and PlGF Values in Different Clinical

Subsets of preeclampsia
In patients with preeclampsia/HELLP, circulating serum concentrations of sFlt-1 were elevated, PlGF levels were decreased, and
the sFlt-1/PlGF ratio was increased when compared to patients
with uneventful pregnancy outcome. The median sFlt-1/PlGF
ratio in all patients with preeclampsia/HELLP was 185 (n=234;

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348HypertensionFebruary 2014
Table 1. Norm Values for the sFlt-1/PlGF Ratio, sFlt-1, and PlGF
Completed Weeks of Gestation
Centiles

1014

1519

2023

2428

2933

3436

37

sFlt-1/PlGF
ratio
Q 2.5

7.39

3.18

1.29

0.88

0.84

0.90

1.67

Q 5

9.27

3.51

1.82

0.95

0.94

1.23

2.18

Q 10

11.6

4.67

2.22

1.22

1.22

2.15

3.81

Median

24.8

10.5

4.92

3.06

3.75

9.03

19.6

Q 90

46.6

20.5

11.0

7.49

16.1

43.4

85.7

Q 95

54.6

25.7

14.6

10.0

33.9

66.4

112

Q 97.5

65.0

28.7

17.5

14.9

62.8

89.9

134

sFlt-1
Q 2.5

586

532

477

520

695

912

1262

Q 5

652

708

572

618

773

992

1533

Q 10

776

844

718

722

967

1220

1899

Median

1328

1355

1299

1355

1742

2552

3485

Q 90

2174

2453

2605

2557

3650

5620

7901

Q 95

2501

2807

2997

3205

5165

7363

9184

Q 97.5

2707

3382

3592

3912

5985

8214

11471

PlGF
Q 2.5

26.8

57.2

106

145

91

68.0

48.9

Q 5

28.8

66.2

119

169

114

78.0

54.4
68.6

Q 10

31.3

80.9

143

200

139

98.2

Median

52.6

135

264

465

471

284

191

Q 90

100

251

500

921

1073

831

620

Q 95

122

289

605

1117

1297

984

862

Q 97.5

136

322

694

1668

1567

1378

1006

246

157

217

346

319

224

176

Serum values of the sFlt-1/PlGF ratio, sFlt-1, and PlGF in patients with normal pregnancy outcome. The median as well as
the centiles (Q) 2.5, 5, 10 and 90, 95, and 97.5 in the 7 gestational age windows sorted by completed weeks of gestation are
displayed. n indicates number of patients; PlGF, placental growth factor; and sFlt-1, soluble fms-like tyrosine kinase-1.

25th75th centile, 92.5427) versus 7.78 (n=468; 25th75th centile, 3.2923.9; P<0.001) in women with uneventful pregnancy
outcome (Figure1A, left). In patients with superimposed preeclampsia, the median sFlt-1/PlGF ratio was 75.4 (n=14; 25th
75th centile, 18.0141); in patients with isolated HELLP, 420
(n=15; 25th75th centile, 192814); in patients with preeclampsia not being severe or complicated by HELLP syndrome, 116
(n=99; 25th75th centile, 51.7202); and in patients with severe
preeclampsia, 350 (n=106; 25th75th centile, 170574; P<0.001
KruskalWallis for subgroup comparison; Figure1A, right).
The median sFlt-1/PlGF ratio in patients with early-onset preeclampsia was 424 (n=100; 25th75th centile, 186717) versus 129
(n=134; 25th75th centile, 59207; P<0.001) in late-onset cases.
Patients with uncomplicated pregnancies 33+6 weeks of gestation
(n=200) had a median sFlt-1/PlGF ratio of 3.68 (25th75th centile,
2.037.50) versus 16.2 (25th75th centile, 6.5037.0; P<0.001) in
control patients 34+0 weeks of gestation (n=268; Figure1B).

Diagnostic Accuracy of sFlt-1, PlGF, and the sFlt-1/

PlGF Ratio in Early- and Late-Onset preeclampsia
In the ROC analysis, sFlt-1/PlGF ratio exhibited a superior performance to the single parameters in diagnosing preeclampsia

(Figure2). For all patients with preeclampsia/HELLP, the

ROC area under curve was 0.94 (95% confidence interval [CI],
0.920.96). For sFlt-1 ROC analysis showed a diagnostic accuracy of 0.92 (95% CI, 0.890.94), whereas PlGF yielded an
area under curve of only 0.91 (95% CI, 0.880.93) (Figure 2).
An optimized cutoff was evaluated based on the ROC analysis. For the whole gestational phase, the optimized cutoff
was found to be 34.1, resulting in a sensitivity of 91% and
a specificity of 83.6%. When analyzing the preeclampsia/
HELLP group divided by early- and late-onset preeclampsia, for early-onset preeclampsia an optimized cutoff of 38.5
resulted in a sensitivity of 93% and a specificity of 97%. For
late-onset preeclampsia, a cutoff of 34.6 resulted in a sensitivity of 88.8% and a specificity of 74.6% (Tables2 and 3).

Definition of Gestational PhaseSpecific

Cutoff Values
Separate cutoffs were determined for the early gestational phase
(20+033+6 weeks of gestation) and for the late gestational
phase (34+0 weeks of gestationdelivery). Moreover, to enhance
the diagnostic accuracy of the sFlt-1/PlGF ratio, for each of the
gestational phases, an equivocal zone between 2 cutoff values

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Verlohren et al Preeclampsia and sFlt-1/PlGF Ratio 349

Figure 1. A, Box-and-whisker plots

showing the distribution of the calculated
soluble fms-like tyrosine kinase-1 (sFlt1)/placental growth factor (PlGF) ratio
of patients with preeclampsia (PE)/
hemolysis, elevated liver enzymes, and
low platelets (HELLP) in 20 to 33+6
weeks of gestations vs gestational
phasematched controls (early
gestational phase) and patients with PE/
HELLP in 34+0 weeks of gestation vs
gestational phasematched controls (late
gestational phase). The boxes represent
the interquartile range, the whiskers
represent the range, and the error bars
represent the median. B, Box-andwhisker plots showing the distribution
of the calculated sFlt-1/PlGF ratio of all
patients (20 weeks of gestationterm)
with PE/HELLP vs controls (non-PE/
HELLP, left) and subgroup analysis of
the PE/HELLP group: superimposed
PE, HELLP without concurrent PE, PE
(not severe, not complicated by HELLP
syndrome), and severe PE (right).
The boxes represent the interquartile
range, the whiskers represent the
range, and the error bars represent the
median. *P<0.001 for the comparison
PE/HELLP vs non-PE/HELLP and
#P<0.001 multigroup comparison PE
subgroups.*P<0.001 where appropriate.
Early phase/onset: 20+0 to 33+6 weeks
of gestation; late phase/onset: >34+0
weeks ofgestation.

was established. For the early gestational phase, the rationale for
choosing the cutoffs was to reach a 95% sensitivity (at the low
cutoff) and 95% specificity (at the high cutoff; Figure3). In this
group, a cutoff of 33 resulted in a sensitivity of 95% and a specificity of 94%, corresponding to an LR of 0.05 (95% CI, 0.020.13)
and an LR+ of 15.8 (95% CI, 9.1327.5). In total numbers, 95
of 100 patients were correctly classified as having preeclampsia, whereas 188 of 200 were correctly diagnosed as not having
preeclampsia. However, in the same early phase, a cutoff of 85
resulted in a sensitivity of 88% and a specificity of 99.5% and
yielded an LR+ of 176 (95% CI, 24.91245) and an LR of 0.12
(95% CI, 0.070.21). All 200 patients bar one were correctly classified as healthy, using the cutoff of 85. In combining the cutoffs
33/85, 95 of 100 preeclamptic patients were correctly classified
and only 5 were incorrectly diagnosed as not being preeclamptic,
whereas only 1 in 100 healthy pregnancies was incorrectly classified as having preeclampsia (Tables2 and 3).
For the late gestational phase, the cutoffs were chosen to
reach a minimum of 95% specificity for the early cases of
late-onset preeclampsia/HELLP (34< 37 weeks of gestation). Here, a cutoff of 33 resulted in a sensitivity of 89.6%, a

specificity of 73.1%, an LR of 0.14 (95% CI, 0.090.24), and

an LR+ 3.33 (95% CI, 2.714.10). However, a cutoff of 110
resulted in a sensitivity of 58.2% and a specificity of 95.5%,
corresponding to an LR+ of 13 (95% CI, 7.3423.0) and an
LR of 0.44 (95% CI, 0.360.54). Therefore, when combining the cutoffs of 33/110, a sensitivity of 89.6% and a specificity of 95.5% were reached (Tables2 and 3).

Discussion
The aim of the study was to provide reference ranges and cutoffs for the clinical use of the sFlt-1/PlGF ratio as an aid in diagnosis of preeclampsia. Previous work from our group as well
as from others has consistently shown that the automated measurement of sFlt-1, PlGF and the calculation of the sFlt-1/PlGF
ratio is a reliable tool for the diagnosis of preeclampsia.8,10,1315
We reported earlier a cutoff of 85 for the sFlt-1/PlGF ratio
as an aid in diagnosis of preeclampsia regardless of the gestational week tested. This preliminary cutoff was based on a case
control cohort of 71 patients with preeclampsia and 280 control
patients.8 In the preliminary analysis, a cutoff of 85 resulted in
a sensitivity of 82% and a specificity of 95% for diagnosing

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350HypertensionFebruary 2014

Figure 2. Receiver operating characteristics curves for the discrimination of healthy and preeclamptic pregnancies by means of the
soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio (left), sFlt-1 (middle), and PlGF (right). The black line
represents all patients, the green line represents patients from the early gestational phase, and the blue line represents patients from the
late gestational phase. On the x axis, false positive/1specificity, and on the y axis, true positive/sensitivity are expressed.

preeclampsia. For the subgroup early-onset preeclampsia, the

same cutoff value (85) for the sFlt-1/PlGF ratio resulted in a sensitivity of 89% and a specificity of 97%. The cutoff was selected
to generate a high specificity in a trade-off with sensitivity of
the test.
After concluding the multicenter casecontrol study, we
were now able to look at a total of 234 patients with preeclampsia and 915 control subjects. In-depth analysis of the
whole study cohort revealed a different performance of the
sFlt-1/PlGF ratio. Now, the single cutoff of 85 resulted in a
sensitivity of 75.6% and a specificity of 95.5%. For the subgroup of early-onset preeclampsia, the single cutoff of 85
resulted in a sensitivity of 88% and a specificity of 99.5%.
We calculated optimized cutoffs based on the ROC analysis. For the whole gestational phase, a single, optimized cutoff
of 34.1 resulted in a sensitivity of 91% and a specificity of
83.6%. Preeclampsia is a heterogeneous disease with marked
differences in presentation and outcome especially between
early-onset (33+6 weeks of gestation) and late-onset disease
(34+0 weeks of gestation). The putatively different underlying pathophysiological mechanisms of the early- and lateonset preeclampsia are reflected by a different performance of
sFlt-1, PlGF, and the sFlt-1/PlGF ratio. We therefore aimed to
adequately reflect the putative 2 diseases of preeclampsia in
defining different cutoffs for the early- and late-onset disease.

Introducing 2 cutoffs for the different disease entities resulted

in a higher accuracy in identifying the disease.

Gestational PhaseDependent Cutoffs Framing an

Equivocal Zone Enhance Diagnostic Accuracy
To accurately classify a patient with early-onset preeclampsia, it is
necessary to have a high sensitivity. The number of false-negative
test results has to be low in order not to miss a diseased patient.
When applying the cutoff of 33 with a sensitivity of 95%, we
would have correctly classified 95 of 100 patients in our cohort.
The associated LR is 0.05. On the contrary, the demand to accurately rule out the disease in patients with unspecific symptoms
requires a high specificity. When applying the upper cutoff of 85,
the specificity of 99.5 would correctly classify 199 of 200 patients
as healthy. The corresponding LR+ is 176. Therefore, the use of
the 2 cutoffs in our setting would result in a correct diagnosis of
all bar 1 as not having preeclampsia and in an incorrect diagnosis
of only 5 of 100 as falsely not having the disease. The equivocal
zone contains 7% of the patients with preeclampsia/HELLP and
5.5% of the controls in the early gestational phase. However, the
equivocal zone for late-onset preeclampsia contains 31.3% of the
preeclampsia/HELLP and 22.4% of the control patients in the
late gestational phase. The establishment of the equivocal zone
allows for the identification of patients at intermediate risk. A
precise diagnosis at the outer borders, combined with a necessity

Table 2. Sensitivities and Specificities With 95% CIs for the Whole as Well as for Early and Late Gestational Phases
Whole Gestational Phase (Wk 20+0Delivery)
Sens/Spec

Absolute

Relative

95% CI

Early Gestational Phase (Wk 20+033+6)

Sens/Spec

Cutoff 85

Absolute

Relative

95% CI

Late Gestational Phase (Wk 34+0Delivery)

Sens/Spec

Absolute

Cutoff 33

Relative

95% CI

Cutoff 33

Sens

177/234

75.6%

70.6100

 Sens

95/100

95.0%

89.8100

 Sens

120/134

89.6%

84.2100

Spec

447/468

95.5%

93.6100

 Spec

188/200

94.0%

90.5100

 Spec

196/268

73.1%

68.3100

Cutoff 85

Cutoff 110

 Sens

88/100

88.0%

81.3100

 Sens

78/134

58.2%

50.7100

 Spec

199/200

99.5%

97.7100

 Spec

256/268

95.5%

92.9100

CI indicates confidence interval; Sens, sensitivities; and Spec, specificities. Bold values indicate the outer borders of the cut-off.

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Verlohren et al Preeclampsia and sFlt-1/PlGF Ratio 351

Table 3. Positive and Negative Likelihood Ratios (LR+/LR)
With 95% CIs for Early and Late Gestational phases
Early Gestational Phase
(Wk 20+033+6)
LR

Estimate

Late Gestational Phase

(Wk 34+0Delivery)

95% CI

LR

Cutoff 33
LR+
LR

15.8
0.05

9.1327.5

LR+

0.020.13

LR

Cutoff 85
LR+
LR

176
0.12

Estimate

95% CI

Cutoff 33
3.33

2.714.10

0.14

0.090.24

Cutoff 110
24.91245

LR+

13

7.3423.0

0.070.21

LR

0.44

0.360.54

CI indicates confidence interval; LR, likelihood ratio. Bold values indicate the
outer borders of the cut-off.

of timely retesting in patients inside the equivocal zone, allows

for maximum diagnostic safety.

Limitations
Our study has limitations. We have performed a casecontrol
study with a 2:1 matching in the ROC group. Because of the
design, with a constructed incidence of preeclampsia of 33.3%,
we were not able to calculate positive or negative predictive values. Individual cutoffs could be established only for 2 gestational
phases. The data set is too small to establish cutoffs for shorter
time intervals, which resulted in a sudden change of cutoffs at
34+0 weeks of gestation. For the late gestational age group,
there is a significant proportion of patients inside the equivocal
zone. Almost 30% of the patients with preeclampsia/HELLP
and almost 25% of the controls need to be retested. For these
patients, data are lacking on when and how often they have to be
retested. We evaluated singleton pregnancies in this study, therefore our results only apply to this group of patients. It is known,
however, that multifetal gestations exhibit different patterns of
angiogenic factor serum levels with healthy twin pregnancies
exhibiting a 3-fold increase in the sFlt-1/PlGF ratio as compared

with singleton pregnancies.16 Therefore, caution must be taken

when applying the reference ranges and cutoffs found in this
study to multifetal gestations. Furthermore, it is noteworthy that
only a small proportion of patients in this European study are of
other than white origin. Therefore, results may vary in populations with a different racial background and thus different disease
prevalence. Further studies are needed to clarify these points.

Clinical Value
The clinical presentation of preeclampsia is diverse, and the
diagnosis of atypical cases of preeclampsia is a daily challenge
to the practicing obstetrician.17 Current definitions and guidelines rely solely on the measurement of blood pressure and proteinuria to diagnose the disease.2,18 However, the measurement
of blood pressure and proteinuria has low predictive accuracy
of adverse maternal and fetal outcomes. Recently, Rana et al9
have shown that in women with suspected preeclampsia, the
sFlt-1/PlGF ratio helps to identify those who will develop a preeclampsia-related pregnancy complication. In addition to the
gold standard of preeclampsia diagnostics, the measurement of
blood pressure and proteinuria, the sFlt-1/PlGF ratio is able to
improve prediction of adverse preeclampsia-related outcomes.9
Various groups have shown that 1 single cutoff at 85 is not
optimal for diagnosing preeclampsia or other hypertensive pregnancy disorders in all clinical settings.14,19,20 The new gestational
phasespecific 2 cutoffs allow maximized accuracy of diagnosis. A more precise diagnosis and gestational phasedependent
analysis might have future therapeutic consequences. Recently,
a pilot study has shown a possible therapy for women with
early-onset preeclampsia. Preeclamptic women who underwent
sFlt-1 apheresis had longer remaining pregnancy duration.21
Removal of sFlt-1 resulted in amelioration of kidney function
and prolongation of pregnancy. Therefore, the sFlt-1/PlGF ratio
might have importance as a parameter to monitor disease severity and hence therapeutic progress.

Figure 3. Gerhard plots displaying the decision limits for ruling in and out as a basis for finding cutoffs and equivocal zone. Values were
taken from receiver operating characteristics analysis: the light blue line represents the sensitivity, and the dark blue line represents the
specificity. Left, The localization of the present cutoff of 85 in the Gerhard plot for the whole gestational phase. Middle, The gestational
phasespecific cutoffs for the early phase. Right, The gestational phasespecific cutoffs for the late phase. The color fills are chosen to
represent interpretations of the test result: below the (lower) cutoff, green indicates low risk; above the (upper) cutoff, red stands for high
risk; inside the equivocal zone, yellow indicates intermediate risk.

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352HypertensionFebruary 2014

Perspectives
The use of an equivocal zone and focus on the outer borders
of this zone to rule in or rule out patients with preeclampsia
enhances the diagnostic accuracy of the sFlt-1/PlGF ratio with
the potential to reduce maternal and fetal morbidity and mortality. Below 34 weeks of gestation, an sFlt-1/PlGF ratio of 33 is
associated with an LR of 0.05, whereas values 85 have a likelihood of a positive test of 176. For late-onset preeclampsia, the
LR at the cutoff 33 is 0.14, the LR+ at 110 is 13. Looking at
the clinical feasibility, a simple diagnostic algorithm is desirable
for the user. However, the establishment of differential cutoffs for
different gestational phases instead of using 1 single cutoff value
better reflects the pathophysiology of the disease. In our opinion,
this approach of gestational phasespecific cutoff values provides
a reasonable balance/compromise between both needs.

Sources of Funding
P. Calda has received funding by grant RVO-VFN64165/2012. The
study was supported by Roche Diagnostics, Germany.

Disclosures
B. Denk is employed by Roche Diagnostics, Penzberg, Germany. H.
Stepan has received consultancy payments from Roche regarding advice on clinical trial design. S. Verlohren has received consultancy
payments from Roche regarding advice on data analysis and publication. S. Verlohren and H. Stepan received lecture fees from Roche.
The other authors report no conflicts.

References
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2. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM.
The classification and diagnosis of the hypertensive disorders of pregnancy:
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3. Verlohren S, Stepan H, Dechend R. Angiogenic growth factors in the diagnosis and prediction of pre-eclampsia. Clin Sci (Lond). 2012;122:4352.
4. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction. BJOG. 2006;113:580589.
5. Soto E, Romero R, Kusanovic JP, Ogge G, Hussein Y, Yeo L, Hassan SS,
Kim CJ, Chaiworapongsa T. Late-onset preeclampsia is associated with
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and without placental lesions consistent with maternal underperfusion. J
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6. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF,
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7. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, Sibai BM,
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8. Verlohren S, Galindo A, Schlembach D, Zeisler H, Herraiz I, Moertl MG,
Pape J, Dudenhausen JW, Denk B, Stepan H. An automated method for
the determination of the sFlt-1/PIGF ratio in the assessment of preeclampsia. Am J Obstet Gynecol. 2010;202:161.e1161.e11.
9. Rana S, Powe CE, Salahuddin S, Verlohren S, Perschel FH, Levine RJ,
Lim KH, Wenger JB, Thadhani R, Karumanchi SA. Angiogenic factors
and the risk of adverse outcomes in women with suspected preeclampsia.
Circulation. 2012;125:911919.
10. Verlohren S, Herraiz I, Lapaire O, Schlembach D, Moertl M, Zeisler
H, Calda P, Holzgreve W, Galindo A, Engels T, Denk B, Stepan H. The
sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders and its prognostic potential in preeclamptic patients. Am J Obstet
Gynecol. 2012;206:58.e158.e8.
11. Report of the National High Blood Pressure Education Program Working
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12. Schneider E, Gleixner A, Hnel R, Leyhe Y, Kleinschmidt C, Beck G,
Steinberg M, Denk B, Gassner D. Technical performance of the first fully
automated assays for soluble fms-like tyrosine kinase 1 and human placental growth factor. Z Geburtsh Neonatol. 2009;213:69.
13. Ohkuchi A, Hirashima C, Suzuki H, Takahashi K, Yoshida M, Matsubara
S, Suzuki M. Evaluation of a new and automated electrochemiluminescence immunoassay for plasma sFlt-1 and PlGF levels in women with
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Fiedler GM, Luthe H, Denk B, Smitz J. Multicenter evaluation of the first
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15. Stepan H, Unversucht A, Wessel N, Faber R. Predictive value of maternal
angiogenic factors in second trimester pregnancies with abnormal uterine
perfusion. Hypertension. 2007;49:818824.
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2011;124:940950.

Novelty and Significance

What Is New?

An imbalance of angiogenic and antiangiogenic factors is involved in the

pathophysiology of preeclampsia.
Automated measurement of antiangiogenic soluble fms-like tyrosine kinase-1 and angiogenic placental growth factor (PlGF) and the calculation
of the soluble fms-like tyrosine kinase-1/placental growth factor ratio
allows for detection of the hypertensive pregnancy disorder.

What Is Relevant?

In the final evaluation of a multicenter study, we show here that the

use of 2 gestational phasespecific cutoffs framing an equivocal zone
improved the diagnostic accuracy of the test.

Summary
The use of multiple cutoffs better reflects the pathophysiological
and clinical difference of early and late preeclampsia and might
thus improve clinical diagnostics and management.

Up to now only 1 cutoff for the clinical use of the soluble fms-like tyrosine
kinase-1/placental growth factor ratio as an aid in diagnosis has been
evaluated.

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Online supplement
HYPE201301787: New gestational phase specific cut-off values for the
use of the sFlt-1/PlGF-ratio as a diagnostic test for preeclampsia

Stefan VERLOHREN, MD1; Ignacio HERRAIZ, MD2; Olav LAPAIRE, MD3;

Dietmar SCHLEMBACH, MD4; Harald ZEISLER, MD5; Pavel CALDA, MD6;
Joan SABRIA, MD7; Filiz MARKFELD-EROL, MD8; Alberto GALINDO, MD2;
Katharina SCHOOFS, MD1; Barbara DENK, PhD9 and Holger STEPAN, MD10

Supplementary Methods:
Patients:
Briefly, hypertension was defined as the repeated measurement of systolic
140 mmHg (Korotkoff I) and diastolic 90 mmHg (Korotkoff V) blood
pressure. Proteinuria was defined as the excretion of 300 mg protein per day
in the 24 hour (h) urine collection or a repeated dipstick of 1+.
PE was defined as severe in contrast to mild, when hypertension 160/110
mmHg, proteinuria 5g/24h or the occurrence of organ failure (kidney, lung) or
neurologic symptoms were observed.
Inclusion criteria for the reference range cohort 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 as stated below.
Samples
Maternal blood was collected by venipuncture in tubes without anticoagulant.
After clotting, the samples were centrifuged with 2000g and serum was
pipetted, and stored at 80C until testing. The sFlt-1 and PIGF
concentrations of each sample were determined in parallel.
Statistics
Gestational age-dependent reference values were determined per time
window as quantiles according to the respective protocols by International
Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and Clinical
and Laboratory Standards Institute (CLSI),Smooth curves for gestational agedependent quantiles were determined using a robust quantile regression
method.

Table S1
Patient Characteristics
Age [y] median (IQR)
Height [cm] median (IQR)
Weight [kg] median (IQR)
BMI [kg/m^2] median (IQR)
Birth Weight [g] median (IQR)
Gestational Week of Delivery median
(IQR)
Max. diastolic BP [mmHg] median
(IQR)
Max. systolic BP [mmHg] median
(IQR)
Smoking: Current [N (%)]
Smoking: Past [N (%)]
Smoking: Never [N (%)]
Smoking: Unknown / NA [N (%)]
Race: White / Caucasian [N (%)]
Race: Black / African American [N (%)]
Race: Other [N (%)]
Race: Unknown / NA [N (%)]
Family History of PE: Yes [N (%)]
Family History of PE: No [N (%)]
Family History of PE: Unknown / NA [N
(%)]
N

PE/HELLP &
20+0-33+6
32.0 (28.0 - 35.0)
164 (160 - 170)
68.0 (56.0 - 81.0)
24.2 (21.5 - 29.9)*
1270 (861 1670)**

controls &
20+0-33+6
31.0 (27.0 - 35.0)
165 (160 - 169)
63.0 (57.0 - 75.0)
23.0 (20.6 - 27.9)
3125 (2779 3490)

PE/HELLP
& >34+0
30.5 (25.0 - 35.0)
165 (160 - 169)
66.5 (60.0 - 77.2)
24.5 (21.6 - 29.0)*
2800 (2200 3291)**

controls &
>34+0
31.0 (27.0 - 34.0)
165 (161 - 170)
64.0 (56.0 - 76.8)
23.5 (20.7 - 27.1)

31.1 (27.9 - 33.2)**

38.6 (37.4 - 39.9)

37.9 (35.9 - 39.4)**

39.4 (38.4 - 40.4)

100 (90.0 110)**

73.0 (70.0 - 80.0)

95.0 (90.0 - 101)**

78.0 (70.0 - 85.0)

160 (149 - 180)**

7 (7%)
14 (14%)
69 (69%)
10 (10%)
82 (82%)
5 (5%)
11 (11%)
2 (2%)
9 (9%)
81 (81%)

120 (110 - 130)

29 (14.5%)
30 (15%)
128 (64%)
13 (6.5%)
173 (86.5%)
5 (2.5%)
20 (10%)
2 (1%)
6 (3%)
163 (81.5%)

150 (142 - 161)**

10 (7.5%)
19 (14.2%)
82 (61.2%)
23 (17.2%)
107 (79.9%)
8 (6%)
13 (9.7%)
6 (4.5%)
7 (5.2%)
105 (78.4%)

123 (113 - 135)

41 (15.3%)
42 (15.7%)
147 (54.9%)
38 (14.2%)
229 (85.4%)
8 (3%)
15 (5.6%)
16 (6%)
9 (3.4%)
228 (85.1%)

10 (10%)
100

31 (15.5%)
200

22 (16.4%)
134

31 (11.6%)
268

3305 (2939 - 3665)

Legend:
Baseline Characteristics: PE/HELLP patients 20+0 33+6 wks and 34+0 wks were compared to gestational age matched controls
(* p<0.05). Data is expressed in median and interquartile range (IQR). BMI = body mass index, BP = blood pressure, NA = not
applicable. IQR (interquartilrange): 25. percentile 75. Percentile, * p<0.05 vs. gestational phase matched controls, **p<0.001
vs. gestational phase matched controls

Figure S1

Figure 1

1149 (2403)
Singleton
pregnancies
915 (2095)
normal pregnancy
outcome

234 (308)
PE/HELLP
outcome

200
early phase

100
early onset

268
late phase

134
late onset

Case-Control Study
1. visit with diagnosis

Case-Control
Study

Reference Range
Study

468
1 visit per patient
matched for gest. wk

38
not normotensive
gest. wk 10

877 (1685)
normotensive, gest. wk 10
multiple visits allowed
but max. 1 visit per window

gest age window 1

gest age window 2
gest age window 3
gest age window 4
gest age window 5
gest age window 6
gest age window 7

Legend:
Study Design: A total of 1149 patients yielding 2403 visits were analyzed in two separate study collectives. For the establishment of
reference ranges 915 singleton pregnancies with normal pregnancy outcome yielding 2095 visits were analyzed. After exclusion of
38 patients due to not being normotensive or yielding samples before wk 10, a total of 877 patients yielding 1685 visits remained.
Analysis was undertaken in seven gestational age windows (gest age window): Window 1: 10+0 - 14+6 week of gestation (wks),
window 2: 15+0 - 19+6 wks, window 3: 20+0 - 23+6 wks, window 4: 24+0 - 28+6 wks, window 5: 29+0 - 33+6 wks, window 6: 34+0
- 36+6 wks and window 7: 37+0 wks delivery. At most one visit from each subject per gestational age window was included in
case of follow-up visits in the control group.
For receiver operating characteristics curves (ROC) and analysis of the cut-off, only visits with a subjects' gestational age of at least
20 weeks were included. A total of 234 subjects with a PE/HELLP-outcome was matched to 468 subjects with uneventful
pregnancy outcome. Legend: early phase / onset: 20+0 33+6 wks; late phase / onset: >34+0 wks.

Figure S2 a

S8 6

Figure S2 b

Figure S2 c

Legend:
Scatter plots of maternal serum concentrations of sFlt-1 (a), PlGF (b) and calculated sFlt-1/PlGF-ratio (c) of uncomplicated
pregnancies (controls). The dots represent individual serum values, the smooth curves represent the median, 2.5 / 5 / 10 as well as
90 / 95 / 97.5 centile of the serum values of sFlt-1, PlGF and the sFlt-1/PlGF-ratio.

New Gestational PhaseSpecific Cutoff Values for the Use of the Soluble fms-Like
Tyrosine Kinase-1/Placental Growth Factor Ratio as a Diagnostic Test for Preeclampsia
Stefan Verlohren, Ignacio Herraiz, Olav Lapaire, Dietmar Schlembach, Harald Zeisler, Pavel
Calda, Joan Sabria, Filiz Markfeld-Erol, Alberto Galindo, Katharina Schoofs, Barbara Denk and
Holger Stepan
Hypertension. 2014;63:346-352; originally published online October 28, 2013;
doi: 10.1161/HYPERTENSIONAHA.113.01787
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563

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World Wide Web at:
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Data Supplement (unedited) at:

http://hyper.ahajournals.org/content/suppl/2013/10/28/HYPERTENSIONAHA.113.01787.DC1.html

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