Establishing New Cut-Off Limits for Galactose 1-Phosphate-Uridyltransferase Deficiency for the Dutch Newborn Screening Programme.

Newborn screening for classical galactosemia in the Netherlands is performed by five laboratories and is based on the measurement of galactose 1-phosphate-uridyltransferase (GALT) activity and total galactose (TGAL) in heel prick blood spots. Unexpected problems with the GALT assay posed a challenge to switch to a new assay. The aim of this study was to make an analytical and clinical evaluation of GALT assays to replace the current assay and to establish new cut-off values (COVs).First, the manual assay from PerkinElmer (NG-1100) and the GSP assay were compared by analyzing 626 anonymous heel prick samples in parallel. Secondly, a manual GSP method was evaluated and 2,052 samples were compared with the automated GSP assay. Finally, a clinical evaluation was performed by collecting data from 93 referred newborns.No satisfactory correlation was observed between GALT activity measured with the manual NG-1100 assay and the automated GSP assay. An acceptable correlation was found between the manual and automated GSP assay. Intra- and inter-assay variation of the automated GSP were 1.8-10.0% and 3.1-13.9%, respectively. Evaluation of clinical data demonstrated that adjusting the COVs for GALT to 2.0 U/dl and TGAL to 1,100 µmol/l improved specificity of screening for classical galactosemia.An assay designed for automated processing to measure GALT activity in heel prick samples works equally well when processed manually. We therefore adopted both methods in the Dutch screening laboratories. As a result of this evaluation new COVs for GALT and TGAL have been introduced and are valid from July 2015.

A pilot study to estimate incidence of guanidinoacetate methyltransferase deficiency in newborns by direct sequencing of the GAMT gene.

BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis causing developmental delays or intellectual disability in untreated patients as a result of irreversible brain damage occurring prior to diagnosis. Normal neurodevelopmental outcome has been reported in patients treated from neonatal period highlighting the importance of early treatment. METHODS: Five hundred anonymized newborns from the National Newborn Screening Program of The Netherlands were included into this pilot study. Direct sequencing of the coding region of the GAMT gene was applied following DNA extraction. The disease causing nature of novel missense variants in the GAMT gene was studied by overexpression studies. GAA and creatine was measured in blood dot spots. RESULTS: We detected two carriers, one with a known common (c.327G>A) and one with a novel mutation (c.297_309dup (p.Arg105Glyfs*) in the GAMT gene. The estimated incidence of GAMT deficiency was 1:250,000. We also detected five novel missense variants. Overexpression of these variants in GAMT deficient fibroblasts did restore GAMT activity and thus all were considered rare, but not disease causing variants including the c.131G>T (p.Arg44Leu) variant. Interestingly, this variant was predicted to be pathogenic by in silico analysis. The variants were included in the Leiden Open Variation Database (LOVD) database (www.LOVD.nl/GAMT). The average GAA level was 1.14µmol/L±0.45 standard deviations. The average creatine level was 408µmol/L±106. The average GAA/creatine ratio was 2.94±0.136. CONCLUSION: The estimated incidence of GAMT deficiency is 1:250,000 newborns based on our pilot study. The newborn screening for GAMT deficiency should be implemented to identify patients at the asymptomatic stage to achieve normal neurodevelopmental outcome for this treatable neurometabolic disease. Biochemical investigations including GAA, creatine and GAMT enzyme activity measurements are essential to confirm the diagnosis of GAMT deficiency. According to availability, all missense variants can be assessed functionally, as in silico prediction analysis of missense variants is not sufficient to confirm the pathogenicity of missense variants.

Dried blood spot measurement of pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit of human chorionic gonadotropin (ß-hCG) from a low-resource setting.

OBJECTIVES: The objectives of the article is to compare pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit of human chorionic gonadotropin (ß-hCG) concentrations in dried blood spots (DBSs) with serum of samples obtained from a public hospital in a low-resource setting and to evaluate their stability. METHODS: Serum and DBS samples were obtained by venipuncture and finger prick from 50 pregnant participants in a cohort study in a public hospital in Accra, Ghana. PAPP-A and ß-hCG concentrations from serum and DBS were measured with an AutoDELFIA® (PerkinElmer, PerkinElmer, Turku, Finland) automatic immunoassay. Correlation and Passing-Bablok regression analyses were performed to compare marker levels. RESULTS: High correlation (>0.9) was observed for PAPP-A and ß-hCG levels between various sampling techniques. The ß-hCG concentration was stable between DBS and serum, PAPP-A concentration consistently lower in DBS. CONCLUSION: Our findings suggest that ß-hCG can be reliably collected from DBS in low-resource tropical settings. The exact conditions of the clinical workflow necessary for reliable PAPP-A measurement in these settings need to be further developed in the future. These findings could have implications for prenatal screening programs feasibility in low-income and middle-income countries, as DBS provides an alternative minimally invasive sampling method, with advantages in sampling technique, stability, logistics, and potential application in low-resource settings.

Relationship between neonatal screening results by HPLC and the number of α-thalassaemia gene mutations; consequences for the cut-off value.

OBJECTIVES: To evaluate the relationship between FAST peak percentage by adapted Bio-Rad Vnbs analysis using the valley-to-valley integration and genotypes with the aim to improve differentiation between severe α-thalassaemia forms (HbH disease) and the milder disease types. METHOD: DNA analysis for α-thalassaemia was performed on 91 dried blood spot samples presenting normal and elevated FAST peak levels, selected during three years of Dutch national newborn screening. RESULTS: Significant differences were found between samples with and without α-thalassaemia mutations, regardless of the genetic profiles. No significant difference was demonstrated between HPLC in -α/αα and -α/-α, between -α/-α and - -/αα and between - -/αα and - -/-α genotypes. CONCLUSION: This study confirms that the percentage HbBart's, as depicted by the FAST peak, is only a relative indication for the number of α genes affected in α-thalassaemia. Based on the data obtained using the modified Bio-Rad Vnbs software, we adopted a cut-off value of 22.5% to discriminate between possible severe α-thalassaemia or HbH disease and other α-thalassaemia phenotypes. Retrospectively, if this cut-off value was utilized during this initial three-year period of neonatal screening, the positive predictive value would have been 0.030 instead of 0.014.

Modeling Down syndrome screening performance using first-trimester serum markers.

OBJECTIVE: To evaluate the modeled predictive value of three current screening markers (pregnancy-associated plasma protein-A (PAPP-A), free ß-human chorionic gonadotropin (free ß-hCG), and nuchal translucency (NT)) and four potential screening markers (a disintegrin and metalloprotease 12 (ADAM12), total hCG, placental protein 13 (PP13), and placental growth factor (PlGF)) for Down syndrome using different screening strategies. METHODS: All markers were measured in stored first-trimester serum of 151 Down syndrome cases and 847 controls. All marker levels were expressed as gestational age-specific multiples of the median (MoMs) and comparisons were made using the Mann-Whitney U-test. Detection rates (DRs) for fixed false-positive rates (FPRs) were modeled using different screening strategies. RESULTS: Significantly different median MoMs for Down syndrome cases compared to controls were found for PAPP-A (0.49 vs. 1.00; P < 0.0001), free ß-hCG (1.70 vs. 1.01; P < 0.0001), ADAM12 (0.89 vs. 1.00; P < 0.0001), total hCG (1.28 vs. 1.00; P < 0.0001), PlGF (0.80 vs. 1.00; P < 0.0001) and NT (1.74 vs. 1.01; P < 0.0001). The lower PP13 MoM in Down syndrome cases (0.91 vs. 1.00) was not statistically significant (P = 0.061). Adding the four new markers to the current screening strategy (i.e. first-trimester combined test) led to an increase in DR from 77% to 80% at a 5% FPR. The modeled application of a two-sample screening strategy (with some markers assessed early and others later in the first trimester) increased the DR to 89%. In a two-step contingent screening model, using an intermediate risk range of 1 in 100 to 1 in 2000 at biochemical screening (using all markers), the overall DR was 77%, but it was predicted that only 33% of women would require referral for NT measurement. CONCLUSIONS: First-trimester Down syndrome screening may be improved by adding new markers to the current screening test and by applying different screening strategies. The application of a two-sample screening model resulted in the highest predicted DR, but this should be confirmed in population-based prospective studies.

Longitudinal trends in fetoplacental biochemical markers, uterine artery pulsatility index and maternal blood pressure during the first trimester of pregnancy.

OBJECTIVE: To assess trends in levels of biochemical markers, uterine artery (UtA) pulsatility index (PI) and maternal blood pressure changes over time and study their relationships in uncomplicated first-trimester pregnancies. METHODS: The study population comprised 86 women with singleton pregnancies. In each woman, a blood sample was collected at 6-7, 8-9, 10-11 and 12-13 weeks' gestation. At the same visit blood pressure was measured and ultrasound examination was performed to measure the crown-rump length and Doppler flow velocity waveform patterns of both UtAs. Serum concentrations of pregnancy-associated plasma protein-A (PAPP-A), free ß-human chorionic gonadotropin (ß-hCG), A disintegrin and metalloprotease domain-containing protein-12 (ADAM-12), placental protein-13 (PP-13) and placental growth factor (PlGF) levels were measured in thawed specimens using an automated time-resolved fluorescence assay. Summary curves were created to describe normal ranges and trends over time. The data were analyzed with a linear mixed model with the log-transformed marker values as dependent variables. This allowed for flexible modeling of patterns over time. RESULTS: Sixty-eight pregnancies had an uneventful outcome, with the birth of an appropriate-for-gestational-age (AGA) infant. In these pregnancies serum PAPP-A, ADAM-12, PP-13 and PlGF levels increased with gestational age. The UtA-PI decreased and the mean arterial blood pressure remained constant. There were no significant correlations between maternal age, birth-weight percentile, gender and blood pressure and any of the biochemical markers. The serum markers were highly correlated with each other except for ß-hCG. A negative correlation was found between most biomarkers and UtA-PI, especially from 10 weeks onwards. Serum concentrations of ADAM-12 and PP-13 were lower in a small-for-gestational-age (SGA) subgroup born at term (n = 6), the former statistically significantly (P = 0.031), the latter non-significantly (P = 0.054), whereas UtA-PI was significantly higher (P = 0.02). Biomarker concentrations in 12 women delivering a large-for-gestational age infant did not differ from those delivering AGA neonates. CONCLUSION: There is a relationship between biochemical markers of early placentation and downstream resistance to flow in the UtAs in low-risk uncomplicated pregnancies, indicating differences in placentation. In a small series of SGA infants born at term we could demonstrate differences as compared to normal pregnancies, with potential value for screening.

Prediction of macrosomia at birth in type-1 and 2 diabetic pregnancies with biomarkers of early placentation.

OBJECTIVE: To evaluate the value of first trimester placental biomarkers (fß-hCG, PAPP-A, ADAM12, PP13 and PlGF) and fetal nuchal translucency (NT) in the prediction of macrosomia at birth in pregestational type-1 and type-2 diabetes (PGDM). DESIGN: Nested case-control study. SETTING: Routine first-trimester combined test. POPULATION: A total of 178 PGDM and 186 control pregnancies. METHODS: ADAM12, PP13 and PlGF concentrations were measured in stored first-trimester serum, previously tested for fß-hCG and PAPP-A. All concentrations were expressed as multiples of the median (MoM). Where applicable, the median MoMs of PGDM and control pregnancies were compared in relation to birthweight centiles (≤90th centile, non-macrosomic, versus >90th centile, macrosomic). Model-predicted detection rates for fixed false-positive rates were obtained for statistically significant markers, separately and in combination. MAIN OUTCOME MEASURES: Prediction of macrosomia in diabetic pregnancies. RESULTS: In the PGDM group, median ADAM12 MoM (0.88; P = 0.007) was lower than in the controls. Subgroup analyses showed that median MoMs of PAPP-A (0.65), ADAM12 (0.85), PP13 (0.81) and PlGF (0.91) were only reduced in the PGDM non-macrosomic birthweight subgroup (n = 93) compared with other weight subgroups. In the PGDM macrosomic birthweight subgroup (n = 69), MoMs of all markers were comparable with the control birthweight subgroups. The screening performance for macrosomia at birth in the PGDM group provided a detection rate of 30% for a 5% false-positive rate (FPR) and 43% for a 10% FPR. CONCLUSIONS: Macrosomia at birth in PGDM pregnancies may be predicted by normal levels of PAPP-A, ADAM12, PP13 and PlGF already in the first trimester of pregnancy. Fetal birthweight in PGDM offspring is partially determined by placental development during the first trimester of pregnancy. The present increase in fetal macrosomia may be related to better early glycemic control and placentation.

First-trimester serum PAPP-A and fß-hCG concentrations and other maternal characteristics to establish logistic regression-based predictive rules for adverse pregnancy outcome.

OBJECTIVE: To determine the clinical relevance of maternal characteristics and first-trimester serum concentrations of pregnancy-associated plasma protein A (PAPP-A) and free beta human chorionic gonadotrophin (fß-hCG) in predicting placenta-related complications, miscarriage and preterm delivery. DESIGN, SETTING AND POPULATION: A historical cohort study of data of the National Institute for Public Health and the Environment of first-trimester screening tests performed between July 2002 and May 2006 was done. Data from 28 566 (64.1%) tests were eligible for analysis. METHODS: By logistic regression, predictive rules were made based on PAPP-A and fß-hCG concentrations, maternal smoking, maternal weight and age, low birth weight, stillbirth and hypertensive disorders, miscarriage and preterm birth. Predictive values were analysed with the area under the curve (AUC) of receiver operating curves (ROC). RESULTS: Predictive for placenta-related complications were low PAPP-A, low fß-hCG, smoking and weight (AUC 54%). For miscarriage low PAPP-A, low fß-hCG and maternal age (MA) were predictive (AUC 78%) and for preterm delivery low PAPP-A, smoking, MA and maternal weight (AUC 55%). CONCLUSION: Only the predictive model for miscarriage had a clinically relevant predictive value of 28%. Results together do not justify closer surveillance of chromosomally normal pregnancies with PAPP-A or fß-hCG levels below the fifth percentile.

First-trimester placental protein 13 and placental growth factor: markers for identification of women destined to develop early-onset pre-eclampsia.

OBJECTIVE: To investigate the predictive value of maternal serum pregnancy-associated plasma protein A (PAPP-A), free ß subunit of human chorionic gonadotrophin (fß-hCG), placental protein 13 (PP13), placental growth factor (PlGF) and a desintegrin and metalloproteinase 12 (ADAM12), for first-trimester identification of early-onset pre-eclampsia. DESIGN: Nested case-control study. SETTING: Routine first-trimester screening for trisomy 21 in the Netherlands. POPULATION: Eighty-eight women who developed pre-eclampsia or haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome before 34 weeks of gestation and 480 controls. METHODS: PP13, PlGF and ADAM12 were measured in stored first-trimester serum, previously tested for PAPP-A and fß-hCG. All marker levels were expressed in multiples of the gestation-specific normal median (MoMs). Model predicted detection rates for fixed false-positive rates were obtained for statistically significant markers alone and in combination. MAIN OUTCOME MEASURES: Development of pre-eclampsia or HELLP syndrome. RESULTS: PP13 and PlGF were reduced in women with pre-eclampsia, with medians 0.68 MoM and 0.73 MoM respectively (P < 0.0001 for both). PAPP-A was reduced (median 0.82 MoM, P < 0.02) whereas ADAM12 and fß-hCG did not differ between control women and those with pre-eclampsia. In pre-eclampsia complicated by a small-for-gestational-age fetus, all markers except fß-hCG had lower values, compared with pregnancies involving fetuses of normal weight. The model-predicted pre-eclampsia detection rate for a combination of PP13 and PlGF was 44% and 54%, respectively, for a fixed 5% and 10% false-positive rate. CONCLUSION: This study demonstrates that PP13 and PlGF in the first-trimester might be promising markers in risk assessment for early pre-eclampsia/HELLP syndrome but for an adequate screening test additional characteristics are necessary.

Proteomics and Down syndrome screening: a validation study.

OBJECTIVE: In a previous discovery study, we identified seven potential screening markers for Down syndrome (DS). Here, we report on an extended study to validate the discriminative potential of these markers. METHODS: Concentrations of the seven analytes were measured using bead-based multiplexed immunoassays in maternal serum from 27 DS pregnancies and 27 matched controls. Control samples were matched to the cases by gestational age (exact day), maternal weight ( ± 5 kg), and maternal age ( ± 1 year) and by closest sample date. Prediction values were obtained for current screening markers [pregnancy-associated plasma protein A (PAPP-A), free beta human chorionic gonadotrophin (fß-hCG) and nuchal translucency (NT)] and seven markers identified before based on concentration fold ratios between DS and controls. Models were fitted based on data of the discovery study or this study and also tested on both datasets. RESULTS: A significantly higher fold ratio was only found for epidermal growth factor (EGF) (-1.96; p = 0.006). In the prediction model for the current dataset, EGF improved the detection rate (DR) of DS by 5.7% [at a fixed 5% false-positive rate (FPR)] when added to the currently used screening markers. CONCLUSIONS: Validation of previously identified biomarkers only confirmed EGF for further consideration as a DS screening marker. This underlines the importance of validating biomarkers; in this study, limiting the range of plausible biomarkers to only one suitable biomarker.

Distributions of current and new first-trimester Down syndrome screening markers in twin pregnancies.

OBJECTIVES: To study the distributions of pregnancy-associated plasma protein A (PAPP-A), the free beta subunit of human chorion gonadotrophin (fbeta-hCG), A Disintegrin and Metalloprotease 12 (ADAM12) and Placental Protein 13 (PP13) in first trimester twin pregnancies. METHODS: Serum marker concentrations were measured in monochorionic and dichorionic twin pregnancies and singleton controls to study differences in multiples of the gestation-specific normal medians (MoMs). RESULTS: Median PAPP-A and fbeta-hCG MoMs were 2.03 and 1.87 for monochorionic twins (n = 116) and 2.18 and 1.89 for dichorionic twins (n = 650). Furthermore, ADAM12 and PP13 MoMs were 1.66 and 1.56 for monochorionic twins (n = 51) and 1.64 and 1.53 for dichorionic twins (n = 249). No statistically significant differences between monochorionic and dichorionic twin pregnancies were found. Correlations between markers in these pregnancies did not differ from singletons. CONCLUSION: For first-trimester screening, different parameters for monochorionic and dichorionic twin pregnancies is not necessary. Furthermore, if ADAM12 and PP13 will be adopted as screening markers, the presented median MoM values, standard deviations and correlation coefficients for twin pregnancies may contribute to a proper twin risk estimation.

Placental protein 13 as a first trimester screening marker for aneuploidy.

OBJECTIVE: To determine whether Placental Protein 13 (PP13) could be an additional marker in first trimester screening for aneuploidies. METHODS: To evaluate differences in multiples of the gestation-specific normal median (MoMs), PP13 concentrations were measured in serum samples from Down syndrome, trisomy 18 and 13 affected pregnancies and euploid singleton pregnancies (four for each case matched for duration of storage, maternal weight and age). RESULTS: The PP13 MoM in Down syndrome cases (n = 153) was 0.91 [not statistically significant from controls (n = 853); P = 0.06; Wilcoxon rank sum test, two-tail]. PP13 MoMs were decreased in trisomy 18 (n = 38-median MoM 0.64; P < 0.0001) and trisomy 13 cases (n = 23-median MoM 0.46; P < 0.0001). There was a slight upward trend in MoM values of the Down syndrome cases with gestational weeks. The PP13 MoM was significantly correlated with the pregnancy associated plasma protein-A MoM and the free beta-subunit of human chorion gonadotrophin (fbeta-hCG) MoM. CONCLUSION: PP13 does not seem to be a good marker for Down syndrome. PP13 MoMs are, however, significantly lower in trisomy 18 and 13 pregnancies. The addition of PP13 to the current screening test could be valuable for improving the discrimination of aneuploid from euploid pregnancies.

Quality of nuchal translucency measurements in The Netherlands: a quantitative analysis.

OBJECTIVE: The Dutch Centre for Population Research has specified quality demands for nuchal translucency (NT) measurement in The Netherlands. We performed an analysis of the quality of NT measurement in 2005-2006 and its influence on screening performance. METHODS: This was a retrospective study of records of NT measurements (n = 27,738) obtained between January 2005 and December 2006 retrieved from the Dutch National Institute for Public Health and the Environment (RIVM). The performance of each individual operator was analyzed with regard to the quality standards, which involved calculation of operator-specific median NT-multiples of the median (MoM) values. For the entire population of operators, a curve was determined describing the relationship between crown-rump length and NT. Detection rates (DR) and false-positive rates (FPR) for Down syndrome were modeled with this new curve and compared to those originally obtained using previously published reference data. RESULTS: Only 22% of all operators met the quality requirement of performing more than 150 NT measurements per year. However, no relationship was found between the number of measurements per operator and their median NT-MoM. The mean of all operator-specific median NT-MoM values was 0.94 (target value 1.0). Overall, operators with The Fetal Medicine Foundation certificate measured a significantly higher median NT-MoM (mean of operator-specific medians, 0.98) as compared to the non-certified operators (0.92). During the study period, the monthly median NT-MoM of all operators rose steadily, from 0.86 in January 2005 to 0.96 in December 2006. Recalculation of the risk for Down syndrome after adjusting the reference NT medians using our own data led to a modeled 4% increase in DR at a 5% FPR. CONCLUSION: Improved monitoring of NT measurement put into effect during the study period seems to have led to an improvement in the accuracy of measurements. Strict quality demands, continued monitoring and scrupulous evaluation of individual operators is likely to lead to an even better performance.

Bead-based multiplexed immunoassays to identify new biomarkers in maternal serum to improve first trimester Down syndrome screening.

OBJECTIVES: To identify new discriminative biomarkers for Down syndrome (DS) pregnancies using a bead-based multiplexed immunoassay, and to use the newly identified biomarkers to construct a prediction model for non-invasive DS screening. METHODS: Maternal serum samples of 14 DS pregnancies and 15 matched controls were analyzed with a bead-based multiplexed immunoassay containing immunoassays for 90 different analytes. Potential biomarkers were selected on the basis of concentration fold ratios between DS and control samples. For these markers and the current screening markers (pregnancy-associated plasma protein-A, PAPP-A; free beta subunit of human chorion gonadotrophin (fbeta-hCG) and nuchal translucency) prediction values were obtained and used to calculate detection rates (DR) at a 5% false positive rate. RESULTS: Seven potential biomarkers of which the fold ratio exceeded 1.3 or -1.3 were selected for further analysis. All 14 DS cases in this study were detected using the combination of all currently used and newly identified markers. The modelled DR for all markers extrapolated to the general pregnant population was 82.5%, compared to a modelled DR of 56.2% for the current screening markers. CONCLUSION: This study demonstrates the possibility of improving the performance of the current first-trimester DS screening by addition of new biomarkers, which were identified using bead-based multiplexed immunoassays.

ADAM12s as a first-trimester screening marker of trisomy.

OBJECTIVE: To evaluate the potential of maternal serum A Disintegrin And Metalloprotease 12-S (ADAM12s) as an additional marker for the combined test in the Dutch first-trimester national Down syndrome (DS) screening program. METHODS: Serum samples were collected between 2004 and 2007 as part of the national program. A total of 218 singleton cases of trisomy 21 (DS), 62 trisomy 18 (Edwards syndrome) and 29 trisomy 13 (Patau syndrome) were identified. All cases were matched with controls for gestation, maternal weight and maternal age. The serum concentration of ADAM12s was determined 'blind' to outcome and expressed in multiples of the gestation-specific median for controls (MoM). RESULTS: The median ADAM12s was 1.00 MoM in controls and in the DS cases at 8, 9, 10, 11, 12, 13 weeks it was 0.45 (n = 3), 0.73 (22), 0.74 (53), 0.85 (37), 0.92 (71), 1.06 (32) MoM, respectively. The median for trisomy 18 was 0.85 MoM and for trisomy 13 0.63 MoM. CONCLUSION: The ADAM12s MoM values were clearly reduced in early first-trimester for all trisomies. However, the screening performance for DS did not greatly improve adding ADAM12s. ADAM12s could be an additional biochemical marker for first-trimester screening for trisomies other than DS.

First-trimester Down syndrome screening performance in the Dutch population; how to achieve further improvement?

OBJECTIVE: To study the performance of the first-trimester combined test between 2004 and 2006 compared to a previous period to investigate changes in time and identify reasons for sub-optimal performance. METHODS: Serum samples were analysed for pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic gonadotrophin (f beta-hCG). Nuchal translucency (NT) was measured between 10 and 14 weeks. Tests were considered screen positive, if their calculated Down syndrome (DS) risk was at least 1 in 250 at term. RESULTS: A total of 20,293 singleton pregnancies were included in the analysis. The median maternal age fell from 35.7 to 34.3 years. The overall median weight-corrected multiple of the median (MoM) values of PAPP-A and f beta-hCG were 1.12 and 1.03, respectively. The median MoM value of NT was 0.89 and increased from 0.82 to 0.96. Sixty-six DS cases were detected by the screening test. The detection rate (DR) for DS was 75.9%, with a FPR of 3.3%. CONCLUSION: The performance of the first-trimester test has improved over the years. A better performance of the NT measurement was the main reason, although NT assessment should further be improved. In addition, a better setting of the medians for the biochemical parameters may contribute to a higher DR.

Fifteen years of triple tests in The Netherlands; the life cycle of a screening test.

OBJECTIVE: This report provides an overview of 15 years prenatal screening for Down syndrome (DS). METHODS: Between 1991 and 2005, blood samples for the triple test were sent for analysis to our laboratory. Test results were considered screen-positive for neural tube defects (NTDs) if the serum alpha-1-fetoprotein > or = 2.50 MoM for singleton pregnancies or screen-positive for DS if the calculated risk was at least 1 in 250. RESULTS: As many as 42 554 tests were performed. Data on the pregnancy outcome were available for 30 290 screening tests (71.2%). In 1991, most requests (93%) came from the university hospitals; thereafter a shift toward midwives occurred. Until 2001, the number of requests rose to 3500 a year. Most samples were collected between 15 and 17 weeks of gestation. The median age of women for whom a test was requested increased from 30.5 to 34.5. The detection rate (DR) for DS remained stable over the years (80%), with a false positive rate of about 13%. The DR for Trisomy 13, 18, and NTD was 50, 68, and 70%, respectively. CONCLUSION: Based on the results of this study, the triple test may be considered a fairly good second trimester screening test. Here it is shown that health practitioners got more acquainted with the test through the years. This may have served the swift introduction of a formal national screening program that started in January 2007.