Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx

Contents lists available at ScienceDirect

Pregnancy Hypertension: An International Journal of

Womens Cardiovascular Health
journal homepage: www.elsevier.com/locate/preghy

Diagnosis of preeclampsia and fetal growth restriction with the

sFlt-1/PlGF ratio: Diagnostic accuracy of the automated immunoassay
Kryptor
Lisa Antonia Drge a,, Alice Hller a, Laura Ehrlich a, Stefan Verlohren a, Wolfgang Henrich a,
Frank Holger Perschel b,c
a
Department of Obstetrics, Charit Universittsmedizin Berlin, Germany
b
Department of Laboratory Medicine, Clinical Chemistry, and Pathobiochemistry, Charit Universittsmedizin Berlin, Germany
c
Labor Berlin Charit Vivantes GmbH, Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Objective: We aimed to characterize the diagnostic accuracy of the Kryptor assay for sFlt-1 and PlGF in
Received 26 November 2016 maternal serum samples of uneventful singleton pregnancies and subjects with preeclampsia (PE) and
Received in revised form 5 February 2017 PE-related outcomes such as fetal growth restriction (FGR). Longitudinal reference ranges of the sFlt-1
Accepted 27 February 2017
and PlGF level in the course of normal pregnancies were generated.
Available online xxxx
Methods: A cohort of subjects with PE and PE-related outcomes including FGR in the third trimester was
compared to a cohort of women with uneventful outcome. Serum levels of sFlt-1, PlGF level as well as the
Keywords:
sFlt-1/PlGF ratio was analysed with the Kryptor assay and compared between the case- and control
Preeclampsia
sFlt-1
groups. Cut-off values were generated and diagnostic accuracy examined.
PlGF Results: Longitudinal reference ranges of the sFlt-1 and PlGF level in healthy pregnancies were in line
with those levels measured with other immunoassays. Comparison of the sFlt-1/PlGF ratio between
PE-related outcomes including FGR or PE and healthy controls showed a high diagnostic accuracy with
an area under the curve (AUC) of 0.917 for PE-related outcomes and 0.919 for PE.
2017 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All
rights reserved.

1. Introduction A precise diagnosis of PE is necessary in order to prevent the

Hypertensive pregnancy disorders such as preeclampsia (PE),
fetal growth restriction (FGR) and HELLP-syndrome (hemolysis,
elevated liver enzymes and low platelet count) are characterized
by an abnormal placentation in the first trimester, leading to
endothelial injury and inflammatory response after 20 weeks of
gestation [1].
Worldwide, 35% of all pregnant women are affected by PE
[2,3]. One per cent of all pregnancies are accompanied by severe
complications such as neurological impairment, severe hyperten-
sion, renal failure, HELLP-Syndrome and coagulopathy [4]. On the
fetal side, a dysfunctional placenta leads to fetal growth restriction
(FGR) and increases the mortality and morbidity rate in 57% of all
newborns worldwide [5].
Corresponding author at: Klinik fr Geburtsmedizin, Charitplatz 1, 10117
Berlin, Germany.
E-mail address: lisa-antonia.droege@charite.de (L.A. Drge).
potentially devastating outcome for both, mother and child [6].
The gold standard, however, is poor in predicting PE-related
adverse outcomes [7].
Increased levels of the anti-angiogenic factor soluble fms-like
tyrosinekinase 1 (sFlt-1) and decreased levels of the angiogenic
placental growth factor (PlGF) are implicated in the pathophysiol-
ogy of placental dysfunction [8]. Automated measurement of these
factors has been implemented as an aid in diagnosis of the disease
[911]. Many clinical studies have shown an elevated sFlt-1/PlGF
ratio in patients with placental dysfunction, especially PE. Cut-off
values of the sFlt-1/PlGF ratio have been established and have been
implemented in the clinical work-up of suspected PE in several
countries [1115].
However, the recently determined cut-off values of the sFlt-1/
PlGF ratio for diagnosis and prediction of PE have been evaluated
with the immunoassay Elecsys by Roche Diagnostics.
The first aim of this study was to generate reference ranges of
sFlt-1, PlGF and the sFlt-1/PlGF ratio measured with the Kryptor
immunoassay (Thermo Fisher Scientific, Henningsdorf, Berlin) in

http://dx.doi.org/10.1016/j.preghy.2017.02.005
2210-7789/ 2017 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: L.A. Drge et al., Diagnosis of preeclampsia and fetal growth restriction with the sFlt-1/PlGF ratio: Diagnostic accuracy of
the automated immunoassay Kryptor, Preg. Hyper: An Int. J. Womens Card. Health (2017), http://dx.doi.org/10.1016/j.preghy.2017.02.005
2 L.A. Drge et al. / Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx
the course of uneventful singleton pregnancies. Second, the sFlt-1/
PlGF ratios were compared between healthy controls and PE
related outcomes. Cut-off values of the sFlt-1/PlGF ratio and their
accuracy to diagnose PE and PE related outcomes were calculated.
2. Materials methods
2.1. Samples and study population
This pregnancy cohort consists of patients that were enrolled in
two clinical studies at the Charit University Hospital. The first
study was performed between September 2007 and June 2010,
the second between March 2013 and July 2014. Both studies inves-
tigated the levels of sFlt-1 and PlGF in patients with and without PE
and/or FGR.
The ethical committee approved these studies and informed
written consents were obtained from all subjects. Stored aliquots
of the study participants were used for this evaluation. The ethical
committee approved the secondary evaluation.
A study population between 18 and 45 years of age was divided
into two arms. In the first arm, we analysed n = 235 out of 265 lon-
gitudinal samples of n = 169 patients with uneventful outcome to
generate normal ranges for the sFlt-1 and PlGF levels and the ratio,
respectively. N = 30 samples were not analysed because of an early
gestational age before 20 weeks of pregnancy.
In the second arm, we compared a cohort with n = 46 patients
with PE related outcomes (PE FGR and FGR) and the n = 33
patients with PE FGR to a gestational age-matched cohort of the
healthy controls (n = 167 in comparison to PE related outcomes
and n = 132 in comparison to PE outcome).
PE was defined according to the guidelines of the International
Society for the Study of Hypertension in Pregnancy (ISSHP) [16].
Hypertension was defined as the repeated measurement of dias-
tolic blood pressure of 90 mmHg and systolic blood pressure
140 mmHg in previously normotensive women. Proteinuria was
diagnosed with 2+ on urine-dipstick of Siemens Healthcare Mul-
tistix Visuals, indicating 100 mg/dl after 60 s as recommended
by the manufacturer, 300 mg protein in a 24-h urine collection or
a spot urine protein/creatinine ratio 30 mg/mmol.
HELLP-syndrome (hemolysis, elevated liver enzymes, low plate-
lets) was defined as increased transaminase (aspartate amino-
transferase, alanine aminotransferase concentrations >2  upper
reference interval), reduced platelet counts (<100.000/ll) plus at
least hemolysis criterion (increased lactate dehydrogenase concen-
trations >2 upper reference interval) or indirect bilirubin serum
concentrations >1,2 mg/dl or reduced haptoglobin serum concen-
trations <0,3 g/l.
FGR was defined as an estimated fetal weight or abdominal cir-
cumference 5th percentile including either an oligohydramnios
with an amniotic fluid index 10th percentile or an increased pul-
satility index of the umbilical artery 95th percentile [17]. All FGR
fetuses additionally fulfilled the criteria of a birth weight 5th per-
centile. Determination of clinical diagnosis and maternal history
was based on information collected at the time of presentation;
follow-up details were documented with the patients report of
delivery in our clinic.
2.2. Samples and assay
In both studies, serum samples were taken at time of diagnosis
or when healthy patients presented for routine visits. The samples
of both studies were collected according to the same standard
operating procedure at our clinic: Maternal blood was collected
by venipuncture without anticoagulant. Samples were then cen-
Please cite this article in press as: L.A. Drge et al., Diagnosis of preeclampsia and fetal growth restriction with the sFlt-1/PlGF ratio: Diagnostic accuracy of
the automated immunoassay Kryptor, Preg. Hyper: An Int. J. Womens Card. Health (2017), http://dx.doi.org/10.1016/j.preghy.2017.02.005
trifuged with 2000g and serum was pipetted and immediately
stored at 80 C until testing.
Measurement of the sFlt-1 and PlGF level was performed on the
fully automated Kryptor Compact Plus system (Kryptor sFlt-1
and Kryptor PlGFplus: Thermo Fisher Scientific, Hennigsdorf, Ber-
lin) according to the manufacturers instructions described else-
where [17]. Assay operators were blinded to clinical information
of the participants. The sFlt-1 assay covers a measuring range of
2290.000 pg/ml. The limit of detection is 22 pg/ml and the limit
of quantization is 34 pg/ml. The PlGF plus-assay covers a measur-
ing range of 3.67000 pg/ml. The limit of detection is 3.6 pg/ml
and the limit of quantization t is 6.9 pg/ml.
2.3. Statistics
All analyses were conducted using the Statistical Package for
Social Sciences 22 (SPSS). Descriptive statistics were computed
for continuous and categorical variables. For continuous variables,
means standard deviation (SD) are given, frequencies and per-
centages are given for categorical data. Median and interquartiles
(IQ) are given for the serum levels of sFlt-1, PlGF and the sFlt-1/
PlGF-ratios. The sFlt-1/PlGF levels of healthy pregnancies of the
longitudinal arm of the studies are presented as 5th, 10th, 50th,
90th and 95th percentiles.
In the second arm of the study, pregnancies with PE related out-
comes such as PE FGR and isolated FGR or the PE FGR group
were matched to the healthy control group. For each patient of
the longitudinal arm, only the last sample in the course of preg-
nancy was used for comparison. Because of different gestational
ages, the total number of the control group varies between both
analyses. However, even though presented in two tables for com-
parison between the PE related outcome group and the PE group,
the control group consists of the same patient cohort.
Test selection was based on the evaluation of the variables for
normal distribution by Kolmogorov-Smirnov test. Given the not
normal distribution of the analyte in both analyses, categorical
data were compared employing the two-tailed exact Fisher test.
Continuous variables were tested employing the Mann-Whitney
test. To generate diagnostic cut-off values for PE and PE related
outcomes when comparing healthy pregnancies to those with
eventful outcome with the sFlt-1/PlGF ratio, ROC curves with Area
under the curve (AUC) were performed. Cut-off values were deter-
mined at the intersection of maximal sensitivity and specificity.
Sensitivities, specificities, positive and negative likelihood were
determined. Raw data were used for calculation of cut-offs and
ROC curves. P values for all tests were two sided and the criterion
for statistical significance was p  0.05, confidence intervals were
set at 95%.
3. Results
3.1. Reference ranges of the sFlt-1/PlGF ratio
The longitudinal ranges for sFlt-1, PlGF and sFlt-1/PlGF of
n = 169 pregnancies with uneventful outcome are presented in
Table 2. N = 41 samples were collected and measured between
2023 weeks of gestation, n = 51 between 24 and 28 weeks,
n = 74 between 29 and 33 weeks, n = 40 between 34 and 36 weeks
and n = 30 between 37 weeks and delivery. Table 1 shows the rel-
atively stable sFlt-1 level until 33 weeks and an increase thereafter.
The decreasing PlGF level in that period leads to a marked increase
of the sFlt-1/PlGF ratio in the third trimester. After 37 weeks, the
value of the sFlt-1/PlGF ratio ranges between 6 at the 5th per-
centile and 388 at the 95th percentile, with a median of 33.
 L.A. Drge et al. / Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx 3

Table 1
Reference ranges of the sFlt-1, PlGF and sFlt-1/PlGF ratio in uneventful pregnancies.

Gestational age Intervals 2023 weeks 2428 weeks 2933 weeks 3436 weeks >37 weeks-delivery
Percentile sFlt-1 pg/ml
P5 418.88 621.26 646.23 788.56 1417.15
P10 587.18 796.16 750.35 1125.30 1726.20
P50 1028.00 1523.00 1501.50 2749.50 3255.00
P90 2257.80 2905.20 3760.50 6258.90 7607.00
P95 4129.40 4195.20 4819.75 7904.35 13159.50
Percentile PlGFpg/ml
P5 83.86 106.54 50.40 12.98 34.30
P10 102.44 158.26 74.75 49.42 41.18
P50 231.50 318.50 277.50 124.30 107.40
P90 472.90 667.74 693.10 421.46 299.00
P95 522.59 803.40 1113.00 584.63 418.73
Percentile sFlt-1/PlGF
P5 1.8 0.92 0.80 2.67 6.01
P10 2.51 1.40 1.37 2.94 8.27
P50 4.66 4.54 6.33 24.32 33.52
P90 9.4 15.80 29.87 117.44 189.59
P95 12.78 30.37 85.54 160.48 388.11
n= 41 51 74 40 30

sFlt-1, PlGF and sFlt-1/PlGF levels in uneventful pregnancies at 2023; 2428; 2933; 3436 weeks and >37 weeks. 265 anylses were performed from n = 165 healthy
subjects. 5th, 10th, 50th, 90th and 95th percentiles are demonstrated. n = Number of visits at the gestational interval.

3.2. Comparison between PE, PE related outcome and controls
When comparing healthy pregnant women with patients with
PE-related adverse outcomes such as FGR and PE FGR, there were
no significant differences in age, BMI, parity, smoking status, eth-
nicity, gestational diabetes and history of PE. However, healthy
pregnant women showed a significantly lower blood pressure,
delivered later, had babies with higher mean birth weight and their
delivery mode was significantly more often spontaneous.
In patients with PE FGR, however, the family history of PE was
more often positive (p = 0.03) as compared to controls. Healthy
controls showed a lower blood pressure, a higher fetal birth weight
and delivered significantly later. The distribution of the delivery
modes between controls and PE cases showed marked differences
(cesarean section rates in 42.4% vs. 54.5%), but they were not sta-
tistically significant. All patients characteristics are presented in
Table 2.
3.3. Angiogenic factor levels in cases and controls
In pregnant women with an uneventful pregnancy outcome, the
levels of sFlt-1 and the sFlt-1/PlGF-ratio increased from week 20
onto delivery. The highest PlGF levels were measured in the gesta-
tional age window 2429 weeks, lowest levels at term (Table 1).
The sFlt-1 and PlGF level, and their ratio respectively, showed
significant differences between patients with PE or PE related out-
comes and healthy controls. The median sFlt-1 level of patients
with PE or PE related outcome was about four times higher when
comparing to the gestational age matched control group
(8643 8566 pg/ml in PE vs. 2238 2164 pg/ml in controls,
p < 0.001 and 8695 7149 pg/ml in PE related outcomes vs.
1921 2026 pg/ml in controls, p < 0.001).
In the PE group, PlGF levels were about five times lower when
comparing to patients with uneventful outcome (40 33 pg/ml in
PE cases vs. 190 282 pg/ml in controls). As compared to controls,
the median PlGF level was about seven times lower in the PE
related outcome group (29 29.24 pg/ml in PE related outcomes
vs. 210 271 pg/ml in controls). Consequently, the sFlt-1/PlGF
ratios were significantly elevated when comparing to healthy con-
trols in both case groups, PE related outcome (286 441 in PE
related outcome vs. 8 21 in controls, p < 0.001) and PE outcome
(286 444 in PE cases vs. 12 32 in controls, p < 0.001). (Table 3
and Fig. 1).
3.4. ROC curves
Receiver Operating Curves (ROC) of the sFlt-1/PlGF ratio for the
diagnosis of PE and PE related outcomes were constructed. ROC
analysis of the sFlt-1/PlGF ratio between patients and controls
yielded an AUC of 0.917 (95% CI 0.8620.972). ROC analysis of
the sFlt-1/PlGF ratio between patients with PE and controls
showed an AUC of 0.919 (0.8560.982, 95% CI).
The established cut-off values to diagnose PE with the Elecsys
immunoassay of Roche Diagnostics of 33, 38 and 85 resulted in a
comparable sensitivity (33 and 38: 90.91%; 85: 87.88%) but in a
decreased specificity (33:73.48%; 38: 75.76%; 85:88.64%, Table 3).
Based on the ROC analysis, we defined an optimal cut-off for
both outcome groups. In the cohort of patients with PE related out-
comes when measuring with the Kryptor assay, an optimal cut-off
of 103 resulted in a sensitivity of 84.78% and a specificity of 93.41%.
In the cohort of patients with PE, the optimal cut-off of 103 leads to
a sensitivity of 87.88% and a specificity of 91.67%.
4. Discussion
We have evaluated the performance of the Kryptor sFlt-1/
PlGF-ratio in a cohort of pregnant women with and without PE.
Reference ranges for these tests and cut-offs for PE and PE related
adverse outcomes were established.
In accordance to other test systems, we were able to show
increasing levels of the sFlt-1 and sFlt-1/PlGF ratio as well as
decreasing levels of PlGF in the course of pregnancy [9]. The med-
ian factor levels measured in healthy pregnant patients are in line
with Salahuddin et al. Between 34 and 36 weeks, they found a
median sFlt-1 level of 2824 pg/ml vs. 2750 pg/ml in our study, a
median PlGF of 177 pg/ml vs. 124 pg/ml and a median sFlt-1/
PlGF ratio 16 vs 24 [18]. Andersen et al. showed comparable values
in healthy singleton pregnancies between 20 and 34 weeks of ges-
tation (mean gestational age 29 weeks, median sFlt-1 level
1066 pg/ml, median PlGF level 343.59 pg/ml, median sFlt-1/PlGF
29) [19].
Next, we investigated Kryptor sFlt-1/PlGF-levels in patients
with PE or PE and PE-related adverse pregnancy outcome. When

Please cite this article in press as: L.A. Drge et al., Diagnosis of preeclampsia and fetal growth restriction with the sFlt-1/PlGF ratio: Diagnostic accuracy of
the automated immunoassay Kryptor, Preg. Hyper: An Int. J. Womens Card. Health (2017), http://dx.doi.org/10.1016/j.preghy.2017.02.005
4 L.A. Drge et al. / Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx

Table 2
Baseline Characteristics and Delivery outcome in uneventful pregnancies and PE related outcomes and PE.

B.1. Controls PE related Outcome p B.2. Controls PE FGR p

Maternal Age (years) 31.33 5.66 30.3 7.83 0.180 31.27 5.21 29.39 6.82 0.137
Maternal Height (cm) 167.32 5.93 165.65 6.40 0.184 167.37 6.03 166.65 6.3 0.765
Maternal Weight (kg) 70.13 18.61 72.89 17.68 0.214 71.55 18.97 76.47 18.98 0.132
Maternal BMI kg/m2 25.08 6.74 26.49 6.32 0.074 25.56 6.78 27.61 6.92 0.088
Nulliparous 80 (47.9%) 27 (58.7%) 0.244 67(50.8%) 20 (60.6%) 0.336
Ethnicity
White/Caucasian 160 (95.8%) 40 (87%) 0.246 125(94.7%) 29 (87.9%) 0.233
Black/Afroamerican 4 (2.4%) 3 (6.5%) 0.173 4(3.0%) 3 (9.1%) 0.144
Asian 3(1.8%) 3 (6.5%) 0.117 3(2.3%) 1 (3.0%) 1.0
Smoking Status
No 154 (92.2%) 44 (95.7%) 0.533 124 (93.9%) 31 (93.9%) 1.0
Yes 11 (6.6%) 1 (2.2%) 0.469 7(5.3%) 1(3.0%) 1.0
Unknown 2 (1.2%) 1 (2.2%) 0.520 1(0.8%) 1(3.0%) 0.361
History of PE
No 146 (87.4%) 39 (94.8%) 0.808 113(85.6%) 26 (78.8%) 0.422
Yes 20 (12.0%) 7 (15.2%) 0.617 18 (13.6%) 7 (21.2%) 0.285
Unknown 1 (0.6%) - 1.0 1(0.8%) - 1.0
Family history of PE
No 131 (78.4%) 31 (67.4%) 0.124 102(77.3%) 25 (75.8%) 0.821
*
Yes 4 (2.4%) 4 (8.7%) 0.068 3 (2.3%) 4 (12.1%) 0.030
Unknown 32 (19.2%) 11 (23.9%) 0.534 27 (20.5%) 4 (21.1%) 0.329
Gestational Diabetes
No 150 (9.8%) 42 (91.3%) 1.0 115(87.1%) 31 (39.9%) 0.370
Yes 17 (10.2%) 3 (6.5%) 0.577 17(12.9%) 2 (6.1%) 0.370
Unknown - 1 (2.2%) 0.216 - -
Delivery mode
*
Vaginal 100 (59.9%) 16 (34.8%) 0.001 76(57.6%) 15 (45.5%) 0.243
*
C-Section 67 (40.1%) 30 (65.2%) 0.001 56(42.4%) 18 (54.5%) 0.243
IUGR - 17 (37%) - 4 (12.1%)
PE - 33 (63%) - 33 (100%)
HELLP - 1 (2.2%) - 1(3.0%)
* *
Fetal birth weight (g) 3055.19 786.78 2177.33 1109.08 0.001 3056.70 766.27 2537.82 1049.48 0.005
* *
GA at delivery (weeks) 37.8 3.2 34.6 4.3 0.001 37.9 2.8 35.6 4.0 0.001
* *
BP systolic 115.78 16.86 142.13 18.76 0.001 118.17 16.19 149.06 14.05 0.001
* *
BP diastolic 72.80 11.63 91.20 14.58 0.001 74.54 11.05 95.30 13.31 0.001
GA at presentation (weeks) 31.1 5.6 32.8 5.1 0.064 232.75 27.94 3.4 4.2 0.053
* *
sFlt-1 pg/ml 1921.00(2026.00) 8695.50 (7149.50) 0.001 2238.00 (2164.00) 8643.00(8565.50) 0.001
* *
PlGF pg/ml 209.90 (270.70) 28.99 (29.24) 0.001 294.01 (37.13) 39.63(32.92) 0.001
* *
sFlt-1/PlGF 8.32 (21.34) 285.97 (440.53) 0.001 40.86 (9.96) 286.19(443.77) 0.001
n= 167 46 132 33

A total of n = 215 subjects were included. Patients with uneventful pregnancy outcome were matched by gestaional age. B1 represents the comparison between the cohort of
a PE related outcome such as FGR or PE FGR and Controls, B2 represents the comparison between the subgroup of PE FGR and Controls. Continuous variables are
demonstrated as Means Standard Deviation. Only the angiogenic factor levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio are presented as Medians and Interquartiles (IQR).
Categorical Data is given as Number (%) Significant differences are indicated where appropriate with *.

Table 3
Cut-off values of the sFlt-1/PlGF ratio A) PE and B) PE related Outcomes.

sFlt1/ Sensitivity (%) CI (95%) Specificity (%) CI (95%) +LR CI (95%) LR CI (95%)
PlGF
Cut-off PE PE related PE PE related PE PE related PE PE related
Outcome Outcome Outcome Outcome
33 90.91 (76.43 89.13 (76.96 73.48 (65.37 79.04 (72.25 3.43 (2.53.4.65) 4.25 (3.125.81) 0.12 (0.04 0.14 (0.060.32)
96.86) 95.27) 80.27) 84.53) 0.36)
38 90.91 (76.43 89.13 (76.96 75.76 (67.79 80.84 (74.20 3.75 (2.725.17) 4.65 (3.356.45) 0.12 (0.04 0.13 (0.060.31)
96.86) 95.27) 82.27) 86.09) 0.36)
85 87.88 (72.67 84.78 (71.78 88.64 (82.10 91.02 (85.71 7.73 (4.72 9.44 (5.74 0.14 (0.05 0.17 (0.080.33)
95.18) 92.43) 92.99) 94.48) 12.66) 15.53) 0.34)
Opt. 87.88 (72.67 84.78 (71.78 91.67 (85.69 93.41 (88.59 10.55 (5.91 12.87 (7.17 0.13 (0.05 0.16 (0.080.32)
103 95.18) 92.43) 95.28) 96.28) 18.83) 23.08) 0.33)

Diagnostic Accuracy for PE FGR (n = 33 vs. n = 132) and PE related Outcome such as FGR or PE FGR (n = 46 vs. n = 167 controls) using the predefined cut-offs of 33, 38 and
85 of the sFlt-1/PlGF ratio for early (less than 34 weeks of gestation and late gestational (34 weeks of gestation)) phases using the Elecsys assay and 103 with best sensitivity
and specificity in the ROC analysis. Sensitivity and specificity in percent (%). +LR: positive likelihood and LR: negative likelihood.

comparing to published results of Verlohren et al., we found
marked differences. The median sFlt-1 concentration in our cohort
of patients with PE was 8643 pg/ml which is comparable to the
median of 12981 pg/mL in the cohort measured on ELECSYS.
The median PlGF value and sFlt-1/PlGF ratio in our cohort of PE
patients were 39.63 pg/mL and 286.19, respectively. This compares
to a PlGF value of 76.06 pg/mL and a sFlt/PlGF ratio of 354.5 in the
Elecsys cohort published by Verlohren et al. [16].
The median sFlt-1 and PlGF levels and the sFlt-1/PlGF ratio of
patients with an adverse outcome were comparable to the results
of Salahuddin et al. in the case group before 34 weeks of gestation
[18]. In the case group after 34 weeks of gestation, we found lower

Please cite this article in press as: L.A. Drge et al., Diagnosis of preeclampsia and fetal growth restriction with the sFlt-1/PlGF ratio: Diagnostic accuracy of
the automated immunoassay Kryptor, Preg. Hyper: An Int. J. Womens Card. Health (2017), http://dx.doi.org/10.1016/j.preghy.2017.02.005
 L.A. Drge et al. / Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx 5

Fig. 1. Angiogenic factors in controls, PE and PE related outcomes. Box-and-whisker plots displaying the logarithmic distribution of the (A) soluble fms-like tyrosine kinase-1
(sFlt-1), (B) placental growth factor (PlGF), and (C) sFlt-1/ PlGF ratio values in subjects with uneventful pregnancy (blue boxes) and I. PE or II. PE related outcomes (red boxes).
Boxes indicate interquartile range; whiskers indicate range; error bars indicate median. Pairwise comparisons among outcome groups were conducted by nonparametric
Mann-Whitney-U testing and are symbolized when significant by black horizontal square brackets, respectively. Asterisks and the according p-value indicate significance.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sFlt-1 levels, a higher PlGF level and a lower consequent sFlt-1/
PlGF ratio. This is explained by a difference in the case groups with
Salahuddin defining an adverse outcome as any event associated to
any hypertensive pregnancy disorder. Andersen et al. comparing
PE cases (n = 39) to controls (n = 76) at a later gestational age of
38 weeks showed a lower median sFlt-1/PlGF ratio of 150 in PE
cases and a higher sFlt-1/PlGF ratio of 48 in controls, leading to a
lower AUC of 0.746 at the mean gestational age of 38 weeks, but
to an almost identical AUC of 0.929 (0.8181.00) in early-onset
PE. With a mean gestational age at presentation of 33 weeks, the
results of the present study are in line with the findings of Ander-
sen et al. [19].
We found an AUC of 0.919 for diagnosing PE which is compara-
ble to the published accuracy of the Elecsys assay (0.97 in early
onset PE and 0.89 in late onset PE) [16]. At the established cut-
off of 85, measurement of the sFlt-1/PlGF-ratio on KRYPTOR
yielded similar accuracy as compared with the Elecsys assay. The
sensitivity was 87.88% and the specificity 87.44% to detect PE as
compared to 82% and 95%. PE-related adverse events were predic-
tied with a sensitivity of 84.78% and specificity 91.02% when mea-
sured on Kryptor. We then determined an optimal cut-off for
detecting PE and/or PE-related adverse outcomes. This cut-off
was found to be 103. At this cut-off, PE and/or adverse outcomes
were detected with a sensitivity of 88% and a specificity of 92%.
Van Helden et al. compared the Elecsys and the Kryptor assay
and found a high correlation for the analytes as well as the ratio
(sFlt-1 r = 0.996, PlGF r = 0.990, sFlt-1/PlGF ratio r = 0.947) [20].
They proposed an optimal cut-off value of 99 of the sFlt-1/PlGF
ratio to diagnose PE with the Kryptor assay that is comparable
to the optimal cut-off of 103 in our study. No diagnostic quality dif-
ferences between the Kryptor and Elecsys assay have been
demonstrated.

Please cite this article in press as: L.A. Drge et al., Diagnosis of preeclampsia and fetal growth restriction with the sFlt-1/PlGF ratio: Diagnostic accuracy of
the automated immunoassay Kryptor, Preg. Hyper: An Int. J. Womens Card. Health (2017), http://dx.doi.org/10.1016/j.preghy.2017.02.005
6 L.A. Drge et al. / Pregnancy Hypertension: An International Journal of Womens Cardiovascular Health xxx (2017) xxxxxx
5. Study limitations
In view of the small case number, we did not distinguish
between either severe or mild PE and early or late-onset PE in
the case groups. The cohort of a PE related outcome was composed
of subjects with either PE with or without HELLP syndrome or FGR.
Only one patient of this analysis was diagnosed with HELLP syn-
drome which underlines the limited validity of the study regarding
the HELLP syndrome. Other hypertensive pregnancy disorders such
as gestational induced hypertension or isolated eclamptic seizures
and placental abruption did not present or occur in time period of
our study.
Differences in median levels of the analytes as well as the ratio
might be explained partially by sample size. Larger prospective
studies are needed, a direct comparison of the diagnostic accuracy
of different assays is desired.
6. Conclusion and outlook
This study generated normal ranges as well as clinical cut-offs
for Kryptor sFlt-1, PlGF and sFlt-1/PlGF ratio in the course of preg-
nancy in healthy pregnant women as well as women with PE or
PE-related adverse outcomes. We found differences in the absolute
values when comparing to published normal ranges of other test
systems. We did not find marked differences in the overall clinical
performance between both test systems, but different cut-off
values lead to a better performance. For the Kryptor system, an
optimal cut-off of 103 is leading to a high clinical sensitivity and
specificity in aiding to diagnose preeclampsia.
Acknowledgement
We would like to acknowledge expert technical assistance of
Gerhild Fiolka performing the analytical assays.
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