NT-proBNP levels in preeclampsia, intrauterine growth restriction as well as in the prediction on an imminent delivery.

OBJECTIVES: Studies of cardiovascular function in pregnancy have shown inconsistent and, in some cases, contradictory results, particularly regarding cardiac output. While some studies report preeclampsia associated with high cardiac output, other studies suggest that preeclampsia should be further subdivided into women with high or low cardiac output. This study was conducted to examine the NT-proBNP levels in preeclampsia, intrauterine growth restriction, and hypertensive pregnancies without preeclampsia. We also examined N-terminal pro-B natriuretic peptide (NT-proBNP) levels three to four months after delivery, in preeclamptic women as well as the prediction of delivery within 10 days. In a reduced number of preeclamptic women and controls we performed echocardiograms to study their diastolic function. METHODS: We investigated the NT-proBNP levels in 213 subjects with preeclampsia only, 73 with intrauterine growth restriction, 44 with preeclampsia and intrauterine growth restriction, 211 who were hypertensive and 662 unaffected pregnancies (controls). We also performed echocardiograms on 36 preeclampsia and 19 controls before delivery and three to five months after delivery. RESULTS: NT-proBNP levels are higher in early onset preeclampsia than in late onset preeclampsia. Intrauterine growth restriction pregnancies showed a NT-proBNP levels similar to hypertensive and unaffected pregnancies. Compared with healthy pregnancies, women with preterm preeclampsia (<37 gestational weeks) had altered left atrial segments. CONCLUSIONS: We observed that NT-proBNP levels are higher in early onset preeclampsia than in late onset. Moreover, diastolic dysfunction is higher in early onset than in late-onset term preeclampsia. An NT-proBNP value >136 pg/mL has a high positive predictive value for an imminent delivery within 10 days.

A more accurate prediction to rule in and rule out pre-eclampsia using the sFlt-1/PlGF ratio and NT-proBNP as biomarkers.

Background The management of potential pre-eclamptic patients using the soluble FMS-like tyrosine kinase 1 (sFlt-1)/ placental growth factor (PlGF) ratio is characterised by frequent false-positive results. Methods A retrospective cohort study was conducted to identify and validate cut-off values, obtained using a machine learning model, for the sFlt-1/PlGF ratio and NT-proBNP that would be predictive of the absence or presence of early-onset pre-eclampsia (PE) in singleton pregnancies presenting at 24 to 33 + 6 weeks of gestation. Results For the development cohort, we defined two sFlt-1/PlGF ratio cut-off values of 23 and 45 to rule out and rule in early-onset PE at any time between 24 and 33 + 6 weeks of gestation. Using an sFlt-1/PlGF ratio cut-off value of 23, the negative predictive value (NPV) for the development of early-onset PE was 100% (95% confidence interval [CI]: 99.5-100). The positive predictive value (PPV) of an sFlt-1/PlGF ratio >45 for a diagnosis of early-onset PE was 49.5% (95% CI: 45.8-55.6). When an NT-proBNP value >174 was combined with an sFlt-1/PlGF ratio >45, the PPV was 86% (95% CI: 79.2-92.6). In the validation cohort, the negative and positive values were very similar to those found for the development cohort. Conclusions An sFlt-1/PlGF ratio <23 rules out early-onset PE between 24 and 33 + 6 weeks of gestation at any time, with an NPV of 100%. An sFlt-1/PlGF ratio >45 with an NT-proBNP value >174 significantly enhances the probability of developing early-onset PE.

Use of the sFlt-1/PlGF ratio to rule out preeclampsia requiring delivery in women with suspected disease. Is the evidence reproducible?

BACKGROUND: Soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio has been proven to predict preeclampsia occurrence. METHODS: Blood samples from 195 pregnant women with suspected preeclampsia were obtained at obstetric triage admission or from the high-risk pregnancy outpatient office. Serum PlGF and sFlt-1 were measured by an electrochemiluminescence immunoassay (ECLIA) on the immunoanalyser Cobas e601 (Roche Diagnostics) and the corresponding ratio was calculated. Final outcomes were reviewed by an independent obstetrician. Only the first determination was considered. RESULTS: A sFlt-1/PlGF ratio of 38 or lower ruled out the need for pregnancy termination due to preeclampsia in the subsequent week with a negative predictive value (NPV) of 99.1% (sensitivity 97.1% and specificity 67.5%). None of the 76 pregnancies with first determination of an sFlt-1/PlGF ratio of 38 or lower between 24 and 34 weeks of gestation delivered due to early-onset preeclampsia. Positive likelihood ratio (PLR) of an sFlt-1/PlGF ratio above 38 for prediction of pregnancy termination due to preeclampsia within 4 weeks is analogous to published evidence. CONCLUSIONS: Between 24 and 34 weeks of gestation, no subsequent determination was needed to completely rule out early-onset preeclampsia when the first sFlt-1/PlGF ratio determination was 38 or lower in singleton pregnancies with signs or symptoms of this syndrome. These findings, if confirmed, will reduce costs and facilitate the implementation of the sFlt-1/PlGF ratio in women with clinical suspicion of preeclampsia in the third trimester.

Role of vitamin D and sFlt-1/PlGF ratio in the development of early- and late-onset preeclampsia.

BACKGROUND: The imbalanced production of placental biomarkers and vitamin D deficiency have been proposed as risk factors for the development of preeclampsia (PE). However, little is known about the relationship between them and their role in early- versus late-onset PE. The objectives were to assess the role of 25-hydroxyvitamin D [25(OH)D] concentrations and the soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio in the development of early- and late-onset PE; and to evaluate the relationship between 25(OH)D and the biomarkers. METHODS: A retrospective, full-blinded cohort study was conducted at the Obstetric Emergency Service of a tertiary care hospital. Pregnant women (n=257) attending obstetric triage with suspicion of PE were included. sFlt-1, PlGF and 25(OH)D concentrations were measured by electrochemoluminescence (ECLIA) immunoassay and pregnancy outcome (development of PE) was registered from patients records. RESULTS: PE women showed lower 25(OH)D concentrations at clinical presentation than non-PE women (median: 35.0 nmol/L and 39.6 nmol/L, respectively; p=0.027). Women with 25(OH)D levels <50 nmol/L experienced an increased risk of developing late-onset PE [odds ratio (OR) 4.6, 95% confidence interval (CI) 1.4-15], but no association was found for early-onset PE. However, a sFlt-1/PlGF ratio above the corresponding cutpoints increased the risk of developing both early- and late-onset PE [ORs 58 (95% CI 11-312) and 12 (95% CI 5.0-27), respectively]. No association was found between 25(OH)D levels and sFlt-1/PlGF ratio. CONCLUSIONS: Low vitamin D status in women with suspected late-onset PE increases the risk of imminent development of the disease.

New biomarkers in diagnosis of early onset preeclampsia and imminent delivery prognosis.

BACKGROUND: Several studies have revealed a high soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio in preeclamptic women. However, its role in patients with suspected preeclampsia (PE) at triage in the emergency department remains an issue and a controversial unique cutpoint of 85 has been proposed regardless of gestational age. A new cutpoint for sFlt-1/PlGF ratio was investigated to rule out PE at obstetric triage, and to assess its prognostic value for risk of imminent delivery. METHODS: Blood samples from 257 pregnant women with suspected PE were obtained at obstetric triage admission. Serum PlGF and sFlt-1 were measured by an electrochemoluminiscence immunoassay (ECLIA) on the immunoanalyzer Cobas e601 (Roche Diagnostics) and the corresponding ratio was calculated. Final outcomes (mainly development of PE) were reviewed and time between clinical presentation and delivery was calculated. RESULTS: The best ratio cutpoint to diagnose PE changed according to gestational age: 23 (92.0% sensitivity, 81.1% specificity) and 45 (83.7% sensitivity, 72.6% specificity) for women <34 and ≥ 34 weeks' gestation, respectively. Furthermore, sFlt-1/PlGF ratio inversely correlated with time elapsed between clinical presentation and delivery, and a cutpoint of 178 could predict complications such as imminent delivery or fetal/neonatal death with a sensitivity of 70.6% and a specificity of 97.8%. CONCLUSIONS: The new cut-off values for the sFlt-1/PlGF ratio adjusted by the gestational age at clinical presentation can be used to rule out PE at obstetric triage and to predict imminent delivery with better accuracy than the cutpoint currently accepted.

Biological variation and prognosis usefulness of new biomarkers in liver transplantation.

BACKGROUND: An observational retrospective study has been conducted, including 52 patients (37 male and 15 female), ranging from 22 to 65 years old, who underwent an orthotopic liver transplantation (OLT) at the Hospital Universitario Central de Asturias (HUCA) between 2007 and 2010. METHODS: The main objective was to evaluate the post-OLT critical complication prognosis usefulness of the precursors of three new biomarkers: mid-regional proadrenomedullin (MR-proADM), carboxy-terminal-proendothelin-1 (CT-ProET-1) and mid-regional proatrial natriuretic peptide (MR-ProANP). As all of them are blood pressure mediators, stress-associated physiological phenomena are expected to affect their expression and secretion, mainly those related to blood circulation. Therefore, as a second goal, the biological variability of the biomarkers has been studied in a set of OLT patients without complications during the first postoperative week. The knowledge of the reference change value of the new biomarkers will be interesting for their correct interpretation in future investigations. The prognostic value of the new biomarkers was also compared to that of procalcitonin (PCT). RESULTS: It has been shown that the basal concentration of the biomarkers is higher in patients that undergo OLT than in the normal population, correlating with the severity of the pathology. The intra-individual biological variation of these biomarkers is similar to other biochemical parameters, the reference change value for OLT patients being 90% for CT-proET-1, 112% for MR-proADM and 127% for MR-proANP. CONCLUSIONS: Multivariate analysis showed that MR-proADM was the best biomarker for the prognosis of severe complications.

Predictive Model for Preeclampsia Combining sFlt-1, PlGF, NT-proBNP, and Uric Acid as Biomarkers.

N-terminal pro-brain natriuretic peptide (NT-proBNP) and uric acid are elevated in pregnancies with preeclampsia (PE). Short-term prediction of PE using angiogenic factors has many false-positive results. Our objective was to validate a machine-learning model (MLM) to predict PE in patients with clinical suspicion, and evaluate if the model performed better than the sFlt-1/PlGF ratio alone. A multicentric cohort study of pregnancies with suspected PE between 24+0 and 36+6 weeks was used. The MLM included six predictors: gestational age, chronic hypertension, sFlt-1, PlGF, NT-proBNP, and uric acid. A total of 936 serum samples from 597 women were included. The PPV of the MLM for PE following 6 weeks was 83.1% (95% CI 78.5−88.2) compared to 72.8% (95% CI 67.4−78.4) for the sFlt-1/PlGF ratio. The specificity of the model was better; 94.9% vs. 91%, respectively. The AUC was significantly improved compared to the ratio alone [0.941 (95% CI 0.926−0.956) vs. 0.901 (95% CI 0.880−0.921), p < 0.05]. For prediction of preterm PE within 1 week, the AUC of the MLM was 0.954 (95% CI 0.937−0.968); significantly greater than the ratio alone [0.914 (95% CI 0.890−0.934), p < 0.01]. To conclude, an MLM combining the sFlt-1/PlGF ratio, NT-proBNP, and uric acid performs better to predict preterm PE compared to the sFlt-1/PlGF ratio alone, potentially increasing clinical precision.

Improvement of a prenatal screening program for trisomy 18 in the first trimester of gestation.

BACKGROUND: The aim of this study was to evaluate population parameters (medians, standard deviations and coefficients of correlation) different from those used by the commercial software Elipse(®) v3.0 (Perkin Elmer) in the calculation of prenatal risk of trisomy 18. Moreover, the truncation limits used for extreme values of free ß-human chorionic gonadotropin (fß-hCG), pregnancy associated plasma protein-A (PAPP-A) and nuchal translucency (NT) were revised. METHODS: A calculation engine for the prenatal risk of trisomy 18 was developed [called FMF (Fetal Medicine Foundation) calculator]. Recently, published population parameters for fß-hCG and PAPP-A as well as new truncation limits were included in this calculator. The patient-specific risks obtained by Elipse(®) v3.0 and FMF calculators, were compared in 18,801 pregnant women, including 13 cases of trisomy 18, four cases of trisomy 13 and one case of triploidy. RESULTS: Using a cut-off point of 1:250, FMF calculator increased the detection rate of trisomy 18 from 62% to 100% with a 0.31% increase in the false-positive rate (FPR). When the detection rate was fixed at 100%, the FPR generated by Elipse v3.0 (1.52%) was significantly higher (p<0.0001) than that generated by the FMF calculator (0.36%). Moreover, an improved detection in cases of trisomy 13 and triploidy was observed. CONCLUSIONS: It is recommended that each laboratory reviews the population parameters and truncation limits used in the risk calculation of trisomy 18, in order to obtain an adequate performance in the screening.

Evaluation of population parameters and mathematical strategies for the calculation of prenatal risk of Down syndrome in the first trimester of pregnancy.

OBJECTIVE: To evaluate the population parameters applied to the calculation of risk for Down syndrome (DS) in the first trimester screening (FTS) and the comparison of performance obtained including or excluding maternal age from the mathematical algorithm. METHODS: Three different calculation engines for prenatal risk of DS were developed on the basis of the population parameters from the Serum, Urine and Ultrasound Screening Study, the Fetal Medicine Foundation, and a combination of both of them. These calculators were evaluated in 14,645 first trimester pregnant women, including 59 DS affected fetuses, comparing their performance with that obtained by our commercial software Elipse® (Perkin Elmer Life and Analytical Sciences, Turku, Finland). Advanced first trimester screening (AFS) strategy was also analyzed, and a hybrid strategy (FTS + AFS) was evaluated. RESULTS: By selecting population parameters from the Serum, Urine and Ultrasound Screening Study, the detection rate increased from 76% (Elipse) to 86% with a small increase in the false positive rate (FPR), from 3.3% to 3.7%, respectively. DS screening performance significantly improved by using the hybrid strategy (AFS in pregnant women under 35 years and FTS in pregnant women over 35 years), with a 92% detection rate (FPR: 3.9%). CONCLUSIONS: In the present study, a new hybrid screening strategy has been proposed to achieve DS detection rates higher than 90%, for a convenient <4% FPR.

Non-invasive fetal RHD genotyping in the first trimester of pregnancy.

BACKGROUND: Hemolytic disease of the fetus and newborn (HDN) is caused primarily by feto-maternal RhD incompatibility. Although all RhD negative pregnant women undergo routine antenatal RhD prophylaxis at 28 weeks of gestation, and following delivery if the newborn is RhD positive, HDN has not been eradicated. Here, we investigated fetal Rhesus D (RHD) genotype in maternal plasma during the first trimester of pregnancy in our area. METHODS: Plasma samples were obtained from 111 RhD negative pregnant women, between 9 and 13 weeks of gestation. DNA from maternal plasma containing cell-free fetal DNA (cffDNA) was analyzed by quantitative PCR (qPCR) to detect RHD exons 5 and 7. A beta-globin (HBB) sequence was quantified to estimate total DNA concentration. qPCR results were compared with newborn RhD determined in cord blood serum. The influence of several gestational parameters on DNA concentration was also analyzed. RESULTS: The specificity and sensitivity of the assay was 93% and 100%, respectively, with 97% diagnostic accuracy. Cell-free DNA concentrations during the first trimester of pregnancy were not affected by the gestational parameters studied (free-beta fraction of human chorionic gonadotropin and pregnancy-associated plasma protein A concentrations, fetal sex, materno-fetal ABO blood group incompatibility, maternal weight and gestational age). CONCLUSIONS: Non-invasive fetal RHD genotyping during the first trimester of pregnancy can be determined with a high specificity, thus representing a valuable tool for improving the management of RhD negative pregnant women. As a high percentage of pregnant women participate in the routine first trimester combined screening program for aneuploidies, the fetal RHD study could be of immediate implementation, since the same blood collection could be used.

New quantitative electrochemiluminescence method (ECLIA) for interleukin-6 (IL-6) measurement.

BACKGROUND: A new interleukin-6 (IL-6) electrochemiluminescent immunoassay (ECLIA, Roche Diagnostics) was evaluated and compared to a previous semiquantitative immunoassay (Milenia Biotec). Reference ranges for cord blood plasma were also calculated. METHODS: The new IL-6 ECLIA test was performed using a Cobas E601 analyzer (Roche Diagnostics). The comparison method (Milenia Biotec) was a semiquantitative lateral flow immunoassay, coupled to a digital image capture system (PicoScan). Healthy at term newborns were recruited to establish reference ranges for IL-6 in cord blood plasma. RESULTS: Total imprecision ranged from 3.7% to 8.0% depending on the IL-6 concentrations. The calculated limit of detection for IL-6 measured by ECLIA was 2.63 pg/mL, almost twice as high as that claimed by the manufacturer (1.5 pg/mL). The linearity of the method was verified to 5000 pg/mL. The IL-6 reference limit, obtained from 148 cord blood samples, was 30 pg/mL (90% confidence interval: 19-48 pg/mL). CONCLUSIONS: The new quantitative ECLIA method showed good reproducibility, linearity and functional sensitivity. Additional clinically based studies are needed to elucidate the usefulness of the IL-6 reference limit, calculated here for the first time by ECLIA in cord blood plasma, to aid in the diagnosis of vertical transmission of sepsis.

Cell-free DNA as a noninvasive acute rejection marker in renal transplantation.

BACKGROUND: Acute rejection (AR) is a key conditioning factor for long-term graft function and survival in renal transplantation patients. The standard care with creatinine measurements and biopsy upon allograft dysfunction implies that AR is usually detected at advanced stages. Rapid noninvasive biomarkers of rejection are needed to improve the management of these patients. We assessed whether total cell-free DNA (tCF-DNA) and donor-derived cell-free DNA (ddCF-DNA) were useful markers for this purpose, both in plasma and in urine. METHODS: Plasma and urine samples from 100 renal transplant recipients were obtained during the first 3 months after transplantation. tCF-DNA and ddCF-DNA were analyzed by quantitative PCR for the HBB (hemoglobin, beta) and the TSPY1 (testis specific protein, Y-linked 1) genes, respectively. We observed 19 episodes of AR, as well as other complications, such as acute tubular necrosis, nephrotoxicity, and infections. RESULTS: Plasma tCF-DNA concentrations increased markedly during AR episodes, often before clinical diagnosis, and returned to reference values after antirejection treatment. A cutoff plasma tCF-DNA concentration of 12 000 genome equivalents/mL correctly classified AR and non-AR episodes in 86% of posttransplantation complications (diagnostic sensitivity, 89%; specificity, 85%). Although similar increases were observed during severe posttransplantation infections, use of the combination of plasma tCF-DNA and procalcitonin (PCT), a specific marker of sepsis, significantly improved the diagnostic specificity (to 98%; 95% CI, 92%-100%), with 97% of the episodes being correctly classified. Use of transrenal DNA and ddCF-DNA concentrations did not add relevant information. CONCLUSIONS: Given that renal biopsy is the gold standard for detecting AR, analysis of both plasma tCF-DNA and PCT could permit a more selective use of this invasive procedure.

Phenotyping and relative quantification of vitamin D binding protein in a paediatric population by using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Adequate concentrations of vitamin D are required to ensure bone health and minimize the incidence of multiple extraskeletal diseases. Although total 25-hydroxyvitamin D (25OHD) remains the recommended biomarker for assessing vitamin D status, it has been speculated that free 25OHD correlates better with clinical outcomes. The calculation of free 25OHD depends on the concentrations of vitamin D binding protein (DBP), the determination of which involves different immunoassays and has led to varying results and conclusions. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and relative quantification of DBP isoforms. METHODS: We used serum samples from healthy children ( n = 79), mainly Caucasian (88%). Proteins were denatured, reduced, alkylated and digested with trypsin. Purified peptides were analysed by LC-MS/MS. The DBP phenotype was established by using the combinations of tryptic peptides associated with each of the three isoforms and one peptide common to all of them to perform relative quantification. The genotyping of volunteers ( n = 7) facilitated verification of the ability of our method to correctly identify the DBP phenotype. RESULTS: The DBP phenotype was correctly established in all samples from volunteers, based on the 100% correlation observed with the genotype. The most common DBP phenotype in Caucasian children was 2/1S (34%) and the rarest 1F/1F (2%). The relative quantification of DBP concentrations did not show statistically significant differences between phenotypes ( P = 0.11). CONCLUSIONS: LC-MS/MS enabled simultaneous phenotyping and relative quantification of DBP, while avoiding the analytical limitations of immunoassays and confirming similar concentrations of DBP in all phenotypes.

Increased serum strontium levels and altered oxidative stress status in early-onset preeclampsia.

BACKGROUND: Correctly distinguishing preeclampsia (PE), gestational hypertension (GH), and intrauterine growth retardation (IUGR) is a challenge for clinicians due to existing similarities. In our previous study, we showed that serum strontium (Sr) levels were elevated in preeclamptic women compared to healthy and GH pregnant women at the end of pregnancy. The main aim of this study was to evaluate Sr and oxidative stress in PE at the time of symptoms onset and before and compare it with IUGR/GH. METHODS: Samples collected at symptoms onset included 77 preeclamptic women and 72 women diagnosed with IUGR/GH divided into two groups according to the gestational extraction week (<34 and ≥ 34). Fifteen patients were also serialized until delivery. Samples collected before symptoms onset included 140 women who developed early-onset PE (E-PE, n = 9), late-onset PE (L-PE, n = 13), IUGR (n = 9), GH (n = 32) and no pathologies (n = 77). Strontium, placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), uric acid (UA), creatinine, lipid peroxidation, and total antioxidant activity (TAA) were measured. RESULTS: Mean Sr, sFlt-1/PIGF ratio, UA, and lipid peroxidation/TAA ratio levels were significantly higher (p = 0.002, <0.0001, <0.0001 and = 0.03, respectively) and estimated glomerular filtration rate (eGFR) and TAA significantly lower (p = 0.0008 and < 0.0001, respectively) in E-PE vs other pathologies when gestational extraction week was <34. There was a significant correlation between Sr and eGFR (r = 0.43, p = 0.02), sFlt-1/PIGF ratio (r = 0.56, p = 0.002), TAA and gestational week of sampling (r = -0.45, p = 0.02) and UA (r = -0.82, p < 0.0001) in the E-PE serial samples. No differences were found in Sr levels before symptoms onset. CONCLUSION: Serum Sr concentration and oxidative status are increased in E-PE when compared to other pathologies at the time of symptoms onset. More studies are needed to elucidate the causes of Sr levels elevation and its role in the pathophysiology of PE.