Ultrasound Obstet Gynecol 2019; 53: 367–375 Published online 4 February 2019 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.19178. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Soluble fms-like tyrosine kinase-1 to placental growth factor ratio: ruling out pre-eclampsia for up to 4 weeks and value of retesting H. ZEISLER1 , E. LLURBA2 , F. J. CHANTRAINE3 , M. VATISH4 , A. C. STAFF5 , M. SENNSTRÖM6 , M. OLOVSSON7 , S. P. BRENNECKE8 , H. STEPAN9 , D. ALLEGRANZA10 , M. SCHOEDL11 , S. GRILL11 , M. HUND10 and S. VERLOHREN12 1 Department of Obstetrics and Gynecology, Medical University Vienna, Vienna, Austria; 2 Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain; 3 University of Liège, CHR de la Citadelle, Liège, Belgium; 4 University of Oxford, Oxford, UK; 5 Oslo University Hospital and University of Oslo, Oslo, Norway; 6 Karolinska University Hospital, Stockholm, Sweden; 7 Uppsala University, Uppsala, Sweden; 8 Department of Obstetrics and Gynaecology, The University of Melbourne and Royal Women’s Hospital, Melbourne, Australia; 9 Leipzig University, Leipzig, Germany; 10 Roche Diagnostics International Ltd, Rotkreuz, Switzerland; 11 Roche Diagnostics GmbH, Penzberg, Germany; 12 Charité –Universitätsmedizin, Berlin, Germany K E Y W O R D S: angiogenic factors; PlGF; pre-eclampsia; PROGNOSIS; retesting; rule out; sFlt-1; sFlt-1/PlGF ABSTRACT post-baseline with high negative predictive values (NPV) of 97.9% and 95.7%, respectively. The onset Objectives The soluble fms-like tyrosine kinase-1 (sFlt-1) of pre-eclampsia within 4 weeks was ruled out with a to placental growth factor (PlGF) ratio is generally high NPV (94.3%) and high sensitivity and speci- elevated some time before and at the clinical onset ficity (66.2% and 83.1%, respectively). Compared with of pre-eclampsia. The PROGNOSIS study validated a women who did not develop pre-eclampsia, those who sFlt-1/PlGF ratio cut-off of ≤ 38 to rule out the onset developed pre-eclampsia had significantly larger median of pre-eclampsia within 1 week of testing in women increases in sFlt-1/PlGF ratio at 2 weeks (Δ, 31.22 vs with suspected disease. The aim of this study was to 1.45; P < 0.001) and at 3 weeks (Δ, 48.97 vs 2.39; assess the predictive value of the sFlt-1/PlGF ratio to rule P < 0.001) after their initial visit. Women who developed out the onset of pre-eclampsia for up to 4 weeks, and to pre-eclampsia and/or adverse fetal outcome compared assess the value of repeat measurements. with those who did not had a significantly greater median Methods This was an exploratory post-hoc analysis of increase in sFlt-1/PlGF ratio over the same period (Δ, data from the PROGNOSIS study performed in pregnant 21.22 vs 1.40; P < 0.001 at 2 weeks; Δ, 34.95 vs 2.30; women aged ≥ 18 years with suspected pre-eclampsia, P < 0.001 at 3 weeks). who were at 24 + 0 to 36 + 6 weeks’ gestation at their first clinic visit. Serum samples were collected at Conclusion The Elecsys® immunoassay sFlt-1/PlGF the first visit and weekly thereafter. sFlt-1 and PlGF ratio can help to rule out the onset of pre-eclampsia levels were measured using Elecsys® sFlt-1 and PlGF for 4 weeks in women with suspected pre-eclampsia. immunoassays. Whether the sFlt-1/PlGF ratio cut-off of © 2018 The Authors. Ultrasound in Obstetrics & ≤ 38 used to rule out the onset of pre-eclampsia within Gynecology published by John Wiley & Sons Ltd on 1 week could predict the absence of pre-eclampsia 2, 3, behalf of the International Society of Ultrasound in and 4 weeks post-baseline was assessed. The value of Obstetrics and Gynecology. repeat sFlt-1/PlGF testing was assessed by examining the difference in sFlt-1/PlGF ratio 2 and 3 weeks after the INTRODUCTION first measurement in women with, and those without, pre-eclampsia or adverse fetal outcome. Pre-eclampsia affects 2–5% of pregnancies and is Results On analysis of 550 women, sFlt-1/PlGF ratio a major cause of perinatal and maternal morbidity ≤ 38 ruled out the onset of pre-eclampsia 2 and 3 weeks and mortality1,2 , and the disease has an unpredictable Correspondence to: Prof. S. Verlohren, Department of Obstetrics, Universitätsmedizin Berlin, Campus Mitte, Charitéplatz 1, D-10117 Berlin, Germany (e-mail: stefan.verlohren@charite.de) Accepted: 6 July 2018 © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd ORIGINAL PAPER on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. 368 Zeisler et al. course, with few specific or sensitive methods for diagnosis Altoetting, Germany). Maternal serum levels of sFlt-1 and prediction1,3,4 . and PlGF were determined using the fully automated Pre-eclampsia is associated with a change in circulating Elecsys® sFlt-1 and Elecsys® PlGF assays on the cobas e maternal levels of pro- and antiangiogenic proteins, electrochemiluminescence immunoassay platform (Roche which have been investigated as biomarkers across Diagnostics GmbH, Mannheim, Germany), and were gestational age5 . For example, the soluble fms-like used to calculate the sFlt-1/PlGF ratio. Neither investiga- tyrosine kinase-1 (sFlt-1) to placental growth factor tor nor participants were informed of the result during the (PlGF) ratio is elevated before and during the clinical study. onset of pre-eclampsia6–8 , so the sFlt-1/PlGF ratio has been established as an aid in the diagnosis and Study objectives/endpoints prediction of pre-eclampsia7,9 . Pre-eclampsia and pro- and anti-angiogenic factor imbalances are indicative of This exploratory post-hoc analysis examined whether wider placental dysfunction10,11 . the sFlt-1/PlGF ratio cut-off of ≤ 38, which has The PROGNOSIS study validated the sFlt-1/PlGF been validated previously for ruling out the onset of ratio for the short-term prediction of pre-eclampsia pre-eclampsia within 1 week, could predict the absence in women preterm (before 37 + 0 weeks’ gestation)9 . of pre-eclampsia/eclampsia/HELLP syndrome (referred to PROGNOSIS identified a sFlt-1/PlGF ratio cut-off of as pre-eclampsia hereafter) for 2, 3 and 4 weeks after ≤ 38 to optimally rule out pre-eclampsia within 1 week, the baseline visit. A second exploratory objective was to with a negative predictive value (NPV) of 99.3% (95% assess the value of repeat sFlt-1/PlGF ratio tests 2 and CI, 97.9–99.9%). PROGNOSIS also reported a positive 3 weeks after Visit 1, by examining correlations between predictive value (PPV) for sFlt-1/PlGF ratio > 38 of changes in sFlt-1/PlGF ratio and the onset of pre-eclampsia 36.7% (95% CI, 28.4–45.7%) to establish the onset and the onset of pre-eclampsia and/or adverse fetal of pre-eclampsia within 4 weeks. A sFlt-1/PlGF ratio outcome within 4 weeks. A combined endpoint included > 38 also identified women at risk for shorter remaining pre-eclampsia and adverse fetal outcome within 4 weeks; duration of pregnancy and at higher risk of preterm combined endpoint status was ‘yes’ if pre-eclampsia delivery (< 37 weeks)12 . was diagnosed within 4 weeks or adverse fetal outcome The aims of this study were to establish whether the occurred within 4 weeks, and ‘no’ if neither pre-eclampsia sFlt-1/PlGF ratio can rule out the onset of pre-eclampsia nor any adverse fetal outcome occurred within 4 weeks. for up to 4 weeks and to determine whether retesting at Adverse fetal outcome was defined as perinatal/fetal death, 2 and 3 weeks post-baseline can further elucidate the risk delivery before 34 weeks’ gestation, intrauterine growth of developing pre-eclampsia. restriction, placental abruption, respiratory distress syndrome, necrotizing enterocolitis or intraventricular hemorrhage. METHODS Study design Analysis populations PROGNOSIS was a prospective observational study car- Of the 1273 women enrolled in PROGNOSIS, 500 ried out at 30 sites across 14 countries; full methodological were included in the development cohort and 550 details have been published elsewhere9,13 . PROGNOSIS in the validation cohort (Figure 1). To calculate NPVs was conducted in accordance with the Guidelines for for the sFlt-1/PlGF ratio cut-off of ≤ 38 to rule out Good Clinical Practice and was funded by Roche Diagnos- pre-eclampsia within 2, 3 and 4 weeks after Visit 1, tics. Pregnant women aged ≥ 18 years who were at 24 + 0 data from women in the PROGNOSIS validation cohort to 36 + 6 weeks’ gestation at the baseline visit (Visit 1) and (n = 550) were used; sensitivity and specificity were suspected to have pre-eclampsia as per protocol-defined also calculated in this population. To calculate delta criteria (Table S1), were enrolled. Exclusion criteria sFlt-1/PlGF ratio values between Visit 1 and Visits 3 included manifest pre-eclampsia or eclampsia, confirmed and 4 (i.e. after 2 and 3 weeks), data were used both diagnosis of HELLP syndrome or treatment with an from women in the PROGNOSIS development cohort investigational medicine within 90 days of enrollment. (n = 500) and from those in the PROGNOSIS validation All participants provided written informed consent. cohort (n = 550) who had measurements available at these time points. To determine whether the delta sFlt-1/PlGF Study assessments ratio values predict pre-eclampsia within 4 weeks, only women whose pre-eclampsia status within 4 weeks was Women were assessed at Visit 1 (baseline visit; first evalua- known were considered. The only visits considered were tion of suspected pre-eclampsia and first sample collected), within 28 days after Visit 1 and when pre-eclampsia Visit 2 (7–9 days from Visit 1) and Visits 3–5 (7 ± 2 days was not diagnosed but still suspected. Visits at which after the previous visit). Serum samples (≥ 2 mL) were pre-eclampsia was diagnosed or present after 28 days were collected according to a standard operating procedure and excluded. Only consecutive visits at which pre-eclampsia were analyzed retrospectively at an independent labora- was still suspected, as per protocol-defined criteria, were tory (Kreiskliniken Altoetting-Burghausen, Zentrallabor, considered. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. sFlt-1/PlGF ratio to rule out pre-eclampsia 369 Statistical analysis log transformed (positive values were transformed as log(x + 1), negative values as –log(–x + 1), and values of Delta sFlt-1/PlGF ratio values were calculated as the difference in sFlt-1/PlGF ratio between Visit 3 or 4 zero remained zero). To evaluate if the risk of developing and Visit 1. In addition to a cut-off of 38, a cut-off pre-eclampsia, according to change in sFlt-1/PlGF ratio, of 85 was used, as a sFlt-1/PlGF ratio of ≥ 85 has was different if women had a baseline sFlt-1/PlGF been associated previously with adverse outcome ratio of ≤ 38 compared with a baseline value of and imminent delivery (within 2 weeks)14 . To compare > 38 to < 85, these subgroups were analyzed separately. delta sFlt-1/PlGF ratio values between groups, an The PROGNOSIS study was powered for the primary exact Wilcoxon–Mann–Whitney test was used. Box analysis9,13 rather than for this exploratory post-hoc plots presenting delta sFlt-1/PlGF ratios were absolute analysis; the results should be interpreted accordingly. Women enrolled in study (n = 1273) Excluded (n = 223): Did not meet inclusion criteria or met exclusion criteria (n = 48) Withdrew consent (n = 11) Multiple pregnancy (n = 78) Lost to follow-up (absence of pre-eclampsia not confirmed after 4 weeks) (n = 52) Visit-1 sample not available or usable (n = 34) Women included in development cohort Women included in validation cohort (n = 500) (n = 550) Pre-eclampsia and/or No pre-eclampsia as per Pre-eclampsia No pre-eclampsia as per HELLP syndrome* protocol (n = 98) protocol (n = 101) (n = 399) (n = 452) Figure 1 Flowchart showing enrolment and outcome of women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts. *Ninety-nine women had pre-eclampsia only, one had pre-eclampsia and HELLP syndrome, and one had HELLP syndrome only. Table 1 Baseline characteristics of women with suspected pre-eclampsia in development and validation cohorts of PROGNOSIS study, according to development of pre-eclampsia as per protocol Development cohort Validation cohort No pre-eclampsia Pre-eclampsia No pre-eclampsia Pre-eclampsia Characteristic (n = 399) (n = 101) (n = 452) (n = 98) Age (years) 32 (27–36) 32 (28–36) 31 (26–36) 32 (25–36) Gestational age (weeks) 31.6 (27.3–34.7) 32.1 (27.7–34.4) 31.4 (27.6–34.3) 31.6 (28.0–34.6) Prepregnancy BMI (kg/m2 ) 27.0 (22.3–32.0) 24.9 (21.5–31.2) 26.1 (22.5–30.6) 26.4 (22.8–29.4) Systolic BP (mmHg) 128 (115–140)* 137 (130–149) 125 (110–137)* 137 (126–146) Diastolic BP (mmHg) 80 (70–90)* 85 (80–94) 78 (70–86)* 90 (80–95) Smoker Past 71 (17.8) 22 (21.8) 105 (23.2) 18 (18.4) Current 60 (15.0) 13 (12.9) 70 (15.5) 9 (9.2) Ethnicity Asian 14 (3.5) 7 (6.9) 24 (5.3) 9 (9.2) Black 26 (6.5) 7 (6.9) 20 (4.4) 8 (8.2) Caucasian 355 (89.0) 87 (86.1) 345 (76.3) 73 (74.5) Other 4 (1.0) 0 (0.0) 63 (13.9) 8 (8.2) Data are given as median (interquartile range) or n (%). *Significantly different compared with those in cohort who developed pre-eclampsia (P < 0.01) (calculated using Mann–Whitney U-test for continuous variables and Fisher’s exact test for categorical variables). BMI, body mass index; BP, blood pressure. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. 370 Zeisler et al. RESULTS within 3 weeks and 71 (12.9%) within 4 weeks; Table S2. A low sFlt-1/PlGF ratio of ≤ 38 ruled out the development Baseline characteristics of pre-eclampsia within 2 and 3 weeks with a high NPV Age, gestational age, prepregnancy body mass index (97.9% and 95.7%, respectively) in women suspected and smoking status were not significantly different to be developing pre-eclampsia before 37 + 0 weeks. between participants who developed clinically confirmed Development of pre-eclampsia within 4 weeks was also pre-eclampsia and those who did not (Table 1). For ruled out using a sFlt-1/PlGF ratio of ≤ 38, with a NPV Visits 3 (2 weeks after Visit 1) and 4 (3 weeks after of 94.3%, and sensitivity and specificity of 66.2% (95% Visit 1), data were available for 652 and 528 participants, CI, 54.0–77.0%) and 83.1% (95% CI, 79.4–86.3%), respectively. The median interval between Visits 1 and 3 respectively (Table 2). was 14 ± 1.45 days and between Visits 1 and 4 it was 21 ± 1.63 days. Ruling out pre-eclampsia using change in sFlt-1/PlGF ratio after 2 and 3 weeks Ruling out pre-eclampsia using baseline sFlt-1/PlGF On analysis conducted in women with available data ratio from both the validation and development cohorts, Of the 550 women in the validation cohort, 98 (17.8%) those who did not develop pre-eclampsia had a developed pre-eclampsia, which was seen in 15 (2.7%) lower median increase in sFlt-1/PlGF ratio after within 1 week, 41 (7.5%) within 2 weeks, 60 (10.9%) 2 weeks (, 1.45 (interquartile range (IQR), –0.12 Table 2 Performance of soluble fms-like tyrosine kinase-1 to placental growth factor ratio cut-off of ≤ 38 to rule out onset of pre-eclampsia/ HELLP syndrome within 1 to 4 weeks after baseline, in 550 women with suspected pre-eclampsia in PROGNOSIS validation cohort Rule out onset of pre-eclampsia within: Parameter 1 week 2 weeks 3 weeks 4 weeks NPV (%) 99.3 (97.9–99.9) 97.9 (96.0–99.0) 95.7 (93.3–97.5) 94.3 (91.7–96.3) PPV (%) 9.4 (4.9–15.8) 25.0 (17.8–33.4) 32.8 (24.8–41.7) 36.7 (28.4–45.7) Sensitivity (%) 80.0 (51.9–95.7) 78.0 (62.4–89.4) 70.0 (56.8–81.2) 66.2 (54.0–77.0) Specificity (%) 78.3 (74.6–81.7) 81.1 (77.5–84.4) 82.4 (78.8–85.7) 83.1 (79.4–86.3) Positive likelihood ratio 3.69 (2.73–4.98) 4.14 (3.25–5.27) 3.99 (3.1–5.14) 3.91 (3.02–5.07) Negative likelihood ratio 0.26 (0.09–0.7) 0.27 (0.15–0.48) 0.36 (0.25–0.54) 0.41 (0.29–0.56) Data in parentheses are 95% CI. NPV, negative predictive value; PPV, positive predictive value. (a) (b) P < 0.001 P < 0.001 500 500 Δ sFlt-1/PlGF ratio (Visit 4 – Visit 1) Δ sFlt-1/PlGF ratio (Visit 3 – Visit 1) 100 100 50 50 10 10 5 5 0 0 –5 –5 –10 –10 –50 –50 No pre-eclampsia Pre-eclampsia No pre-eclampsia Pre-eclampsia (n = 610) (n = 42) (n = 518) (n = 10) Figure 2 Change in soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio 2 (a) and 3 (b) weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts, according to development of pre-eclampsia. Boxes represent median and interquartile range and whiskers represent range excluding outliers more than 1.5 × interquartile range from upper or lower quartile. y-axis is in log scale. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. sFlt-1/PlGF ratio to rule out pre-eclampsia 371 to 9.41) (n = 610; Figure 2a)) and after 3 weeks (, (n = 42) (P < 0.001) (Figure 2a)) and after 3 weeks 2.39 (IQR, 0.04–12.49) (n = 518; Figure 2b)). In (, 48.97 (IQR, 25.01–69.67) (n = 10) (P < 0.001) contrast, in women who developed pre-eclampsia, the (Figure 2b)). median difference in sFlt-1/PlGF ratio was significantly For women who had a sFlt-1/PlGF ratio of ≤ 38 at higher after 2 weeks (, 31.22 (IQR, 6.48–62.36) Visit 1 and who did not develop pre-eclampsia, the median (a) (b) 1000 500 P < 0.001 500 P = 0.082 Δ sFlt-1/PlGF ratio (Visit 3 – Visit 1) Δ sFlt-1/PlGF ratio (Visit 3 – Visit 1) 100 100 50 50 10 10 5 5 0 0 –5 –5 –10 –10 No pre-eclampsia Pre-eclampsia No pre-eclampsia Pre-eclampsia (n = 532) (n = 18) (n = 47) (n = 13) Figure 3 Change in soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio 2 weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts, in those with baseline sFlt-1/PlGF ratio ≤ 38 (a) and in those with baseline sFlt-1/PlGF ratio > 38 to < 85 (b), according to development of pre-eclampsia. Boxes represent median and interquartile range and whiskers represent range excluding outliers more than 1.5 × interquartile range from upper or lower quartile. y-axis is in log scale. (a) (b) 500 100 100 Δ sFlt-1/PlGF ratio (Visit 4 – Visit 1) 50 Δ sFlt-1/PlGF ratio (Visit 4 – Visit 1) 50 10 10 5 5 0 0 –5 –5 –10 –10 –50 No pre-eclampsia Pre-eclampsia No pre-eclampsia Pre-eclampsia (n = 472) (n = 5) (n = 25) (n = 2) Figure 4 Change in soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio 3 weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts, in those with baseline sFlt-1/PlGF ratio ≤ 38 (a) and in those with baseline sFlt-1/PlGF ratio > 38 to < 85 (b), according to development of pre-eclampsia. Boxes represent median and interquartile range and whiskers represent range excluding outliers more than 1.5 × interquartile range from upper or lower quartile. y-axis is in log scale. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. 372 Zeisler et al. (a) (b) P < 0.001 P < 0.001 500 500 Δ sFlt-1/PlGF ratio (Visit 4 – Visit 1) Δ sFlt-1/PlGF ratio (Visit 3 – Visit 1) 100 100 50 50 10 10 5 5 0 0 –5 –5 –10 –10 –50 –50 No Yes No Yes (n = 569) (n = 75) (n = 494) (n = 29) Combined endpoint Combined endpoint Figure 5 Change in soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio 2 (a) and 3 (b) weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts, according to development of pre-eclampsia and/or adverse fetal outcome (combined endpoint). Boxes represent median and interquartile range and whiskers represent range excluding outliers more than 1.5 × interquartile range from upper or lower quartile. y-axis is in log scale. difference in sFlt-1/PlGF ratio was , 1.12 (IQR, –0.11 4 weeks, with a median delta sFlt-1/PlGF ratio of 68.9 to 6.94) after 2 weeks (n = 532) (Figure 3a) and , 2.10 (Table S3). (IQR, 0.05–10.48) after 3 weeks (n = 472) (Figure 4a). In comparison, women who developed pre-eclampsia had a higher median increase in sFlt-1/PlGF ratio Maternal and adverse fetal outcomes after 2 weeks (, 20.30 (IQR, 6.48–46.21) (n = 18) The median difference in sFlt-1/PlGF ratio after 2 weeks (P < 0.001) (Figure 3a)) and after 3 weeks (, 34.95 in women who had not developed pre-eclampsia or (IQR, 9.26–68.93) (n = 5) (sample size too low for adverse fetal outcome was , 1.40 (IQR, –0.11 to 8.04) statistical analysis) (Figure 4a)). (n = 569). In comparison, in women who developed Women who had an initial sFlt-1/PlGF ratio of pre-eclampsia or adverse fetal outcome, the median > 38 to < 85 and who did not develop pre-eclampsia increase in sFlt-1/PlGF ratio after 2 weeks was greater, had a median increase in sFlt-1/PlGF ratio of , 19.77 at , 21.22 (IQR, 1.20–49.81) (n = 75) (P < 0.001) (IQR, 2.16–43.84) (n = 47) after 2 weeks (Figure 3b) (Figure 5a). The median increase in sFlt-1/PlGF ratio and , 23.84 (IQR, –2.77 to 74.56) (n = 25) after in women with suspected pre-eclampsia after 3 weeks 3 weeks (Figure 4b). In contrast, those who developed who had not developed pre-eclampsia or adverse fetal pre-eclampsia had a higher median increase in sFlt-1/PlGF outcome was , 2.30 (IQR, 0.01–11.79) (n = 494). In ratio after 2 weeks (, 35.80 (IQR, 22.87–65.54) (n = 13) women who developed pre-eclampsia or adverse fetal (P = 0.082) (Figure 3b)) and after 3 weeks (, 45.81 outcome, the median increase in sFlt-1/PlGF ratio after (IQR, 33.75–57.86) (n = 2) (sample size too low for 3 weeks was greater, at , 34.95 (IQR, 4.94–74.56) statistical analysis) (Figure 4b)). The trend towards a (n = 29) (P < 0.001) (Figure 5b). After both 2 and greater difference in sFlt-1/PlGF ratio between visits in 3 weeks, greater increases in median sFlt-1/PlGF ratio women who developed pre-eclampsia vs those who did were observed in women who developed pre-eclampsia or not, was less pronounced in this subgroup (baseline adverse fetal outcome and had a sFlt-1/PlGF ratio of ≤ 38 sFlt-1/PlGF ratio of > 38 to < 85) than in women who at Visit 1 compared with those who did not develop initially had a sFlt-1/PlGF ratio of ≤ 38. pre-eclampsia or adverse fetal outcome (Figures S1 Of the 64 women who had a sFlt-1/PlGF ratio of ≤ 38 and S2). at Visit 1 (baseline) and a sFlt-1/PlGF ratio > 38 at Visit PlGF and sFlt-1 biomarker levels were analyzed 3 (after 2 weeks), nine developed pre-eclampsia within individually (Table S4). Changes in the sFlt-1 level were 4 weeks and had a median delta sFlt-1/PlGF ratio of 49.5 significantly different between women who developed (Table S3). Of the 69 women with a sFlt-1/PlGF ratio pre-eclampsia and those who did not. In contrast, changes of ≤ 38 at Visit 1 and a sFlt-1/PlGF ratio > 38 at Visit in PlGF level did not significantly differ between the 4 (after 3 weeks), three developed pre-eclampsia within groups. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. sFlt-1/PlGF ratio to rule out pre-eclampsia 373 DISCUSSION previous findings that sFlt-1 increases significantly in early-onset pre-eclampsia, compared with PlGF, which The sFlt-1/PlGF ratio cut-off of ≤ 38 was validated pre- does not decrease significantly as early-onset disease viously to rule out the onset of pre-eclampsia within progresses16 . 1 week in women suspected to have the syndrome, Previous studies have observed a strong link between when tested before 37 + 0 weeks’ gestation9 . The present sequential changes in sFlt-1/PlGF ratio and the devel- post-hoc analysis shows that a sFlt-1/PlGF ratio of opment of pre-eclampsia5,14–18 . These studies, alongside ≤ 38 has a high NPV to rule out pre-eclampsia for our data, suggest that there is value in remeasuring the up to 4 weeks after the initial test, with high sensi- sFlt-1/PlGF ratio in women with suspected pre-eclampsia, tivity and specificity. The ability to rule out devel- opment of pre-eclampsia/eclampsia/HELLP syndrome especially in women with a sFlt-1/PlGF ratio between 38 within the following 4 weeks of pregnancy could help and 85 or 110. clinicians make informed decisions regarding patient Current UK National Institute for Health and Care monitoring. Excellence guidelines recommend that the Elecsys® sFlt-1/PlGF levels are dynamic6,9,11 ; thus, we hypoth- immunoassay sFlt-1/PlGF ratio, alongside standard clin- esized that, in women with suspected pre-eclampsia ical assessment and follow-up, can be used to help who initially had a sFlt-1/PlGF ratio of > 38 to < 85, rule out pre-eclampsia in preterm women presenting an increase in sFlt-1/PlGF ratio over the following 2 with symptoms of the condition19 . Our study suggests or 3 weeks might indicate impending pre-eclampsia or a that the initially recommended 1-week rule-out period higher risk of developing it. There was a significantly could be confidently extended to at least 2 and up to greater increase in the sFlt-1/PlGF ratio between Visit 1 4 weeks, albeit with slightly lower confidence depending and a visit 2 or 3 weeks later in women who devel- on the clinical presentation of the patient. For women oped pre-eclampsia and/or adverse fetal outcome within with suspected pre-eclampsia and a sFlt-1/PlGF ratio 4 weeks, compared with those who did not develop of > 38 to < 85, Stepan et al.20 recommend considering pre-eclampsia and/or adverse fetal outcome. This was a follow-up test after 1 or 2 weeks, as these women most apparent in women with a sFlt-1/PlGF ratio of do not have a definite diagnosis of pre-eclampsia but ≤ 38 at Visit 1, compared with those with a ratio of are highly likely to develop it. Our study suggests that > 38 to < 85 at Visit 1, possibly owing to a higher starting identifying changes in the sFlt-1/PlGF ratio by retesting level meaning there was less scope for increase. Among can stratify a patient’s risk and help guide clinicians women with a sFlt-1/PlGF ratio of ≤ 38 at Visit 1, in the management of at-risk groups. Currently, the nine had developed pre-eclampsia at Visit 3 and three rate of retesting may be low; in a study of real-world had developed pre-eclampsia at Visit 4; these women decision-making based on the sFlt-1/PlGF ratio test21 , the were ruled in with a sFlt-1/PlGF ratio > 38 following actual frequency of retesting was only 6.5%22 . Increas- retesting after 2–3 weeks, demonstrating the value of ing the retesting rate may help identify additional at-risk retesting. women. The prevalence of pre-eclampsia in the PROGNOSIS The clinical presentation of pre-eclampsia is diverse study was relatively low (2.7% within 1 week; 12.9% and the diagnosis of atypical cases of pre-eclampsia is within 4 weeks; 17.8% overall during pregnancy in a daily challenge for obstetricians. The data presented the validation cohort) compared with in previous here highlight the value of the sFlt-1/PlGF ratio in ruling studies; Sabrià et al.15 observed that 33.3% of pregnant out pre-eclampsia for up to 4 weeks in women with women developed pre-eclampsia or HELLP syndrome. suspected pre-eclampsia. Improved triage of patients with The predictive performance of the sFlt-1/PlGF ratio suspected pre-eclampsia will ensure appropriate treatment may vary between target populations with differing and reduce unnecessary hospitalization. prevalences of the disease. With a higher prevalence of The strengths of this study include the large sample size, pre-eclampsia, NPV will be slightly lower and PPV slightly higher. well-characterized participants and the use of prospective Assessment of the change in sFlt-1/PlGF ratio between longitudinal sequential sampling at defined time intervals. visits can provide further information to clinicians, The limitations include potential variation in assay cut-off allowing additional confidence in decision-making. These value from that used in the current study, sample size being findings are in agreement with those reported in a general based on power calculations for the primary PROGNOSIS population of pregnant women, in which women with analysis rather than for this exploratory post-hoc analysis, an increase in sFlt-1/PlGF ratio between the first and small sample sizes used for some retesting analyses, lack second trimesters had a higher risk of developing preterm of data on the need for retesting if the initial test score is pre-eclampsia5 . ≥ 85 and, finally, the inclusion of patients who may not be The finding that the sFlt-1 level increases signif- representative of the general population. This study does icantly in women developing pre-eclampsia in the not validate the optimal time interval for retesting, nor short term, compared with those who do not develop does it assess the predictive performance of the sFlt-1/PlGF pre-eclampsia, suggests that the increase in sFlt-1 ratio across target populations with differing prevalences level reflects the development of imminent disease, in of pre-eclampsia; these should be the objectives of future contrast to PlGF. These results are consistent with studies. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. 374 Zeisler et al. In conclusion, this post-hoc analysis demonstrates the REFERENCES additional value of the Elecsys® sFlt-1/PlGF ratio for the 1. Ananth CV, Keyes KM, Wapner RJ. 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Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. sFlt-1/PlGF ratio to rule out pre-eclampsia 375 SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Figure S1 Change in sFlt-1/PlGF ratio 2 weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts with sFlt-1/PlGF ratio ≤ 38 at baseline (a) and those with sFlt-1/PlGF ratio > 38 to < 85 at baseline (b), according to development of pre-eclampsia and/or adverse fetal outcome. Figure S2 Change in sFlt-1/PlGF ratio 3 weeks after baseline measurement in women with suspected pre-eclampsia in PROGNOSIS development and validation cohorts with sFlt-1/PlGF ratio ≤ 38 at baseline (a) and those with sFlt-1/PlGF ratio > 38 to < 85 at baseline (b), according to development of pre-eclampsia and/or adverse fetal outcome. Table S1 Criteria for suspected pre-eclampsia. Adapted from Hund et al., 201413 Table S2 Contingency table for performance of sFlt-1/PlGF ratio for ruling out onset of pre-eclampsia within 1 to 4 weeks from baseline visit in women with suspected pre-eclampsia Table S3 Categorization of women with suspected pre-eclampsia, based on sFlt-1/PlGF ratio cut-off value at baseline vs after 2 and 3 weeks Table S4 Analysis of change in sFlt-1 and PlGF as single biomarkers between visits and relation to occurrence of pre-eclampsia and/or adverse fetal outcome within 4 weeks from baseline visit in women with suspected pre-eclampsia © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2019; 53: 367–375. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.