Current State and Innovations in Newborn Screening: Continuing to Do Good and Avoid Harm.

In 1963, Robert Guthrie's pioneering work developing a bacterial inhibition assay to measure phenylalanine in dried blood spots, provided the means for whole-population screening to detect phenylketonuria in the USA. In the following decades, NBS became firmly established as a part of public health in developed countries. Technological advances allowed for the addition of new disorders into routine programmes and thereby resulted in a paradigm shift. Today, technological advances in immunological methods, tandem mass spectrometry, PCR techniques, DNA sequencing for mutational variant analysis, ultra-high performance liquid chromatography (UPLC), iso-electric focusing, and digital microfluidics are employed in the NBS laboratory to detect more than 60 disorders. In this review, we will provide the current state of methodological advances that have been introduced into NBS. Particularly, 'second-tier' methods have significantly improved both the specificity and sensitivity of testing. We will also present how proteomic and metabolomic techniques can potentially improve screening strategies to reduce the number of false-positive results and improve the prediction of pathogenicity. Additionally, we discuss the application of complex, multiparameter statistical procedures that use large datasets and statistical algorithms to improve the predictive outcomes of tests. Future developments, utilizing genomic techniques, are also likely to play an increasingly important role, possibly combined with artificial intelligence (AI)-driven software. We will consider the balance required to harness the potential of these new advances whilst maintaining the benefits and reducing the risks for harm associated with all screening.

Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

Prediction of VLCAD deficiency phenotype by a metabolic fingerprint in newborn screening bloodspots.

PURPOSE: Newborns who test positive for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening may have a severe phenotype with early onset of life-threatening symptoms but may also have an attenuated phenotype and never become symptomatic. The objective of this study is to investigate whether metabolomic profiles in dried bloodspots (DBS) of newborns allow early phenotypic prediction, permitting tailored treatment and follow-up. METHODS: A metabolic fingerprint was generated by direct infusion high resolution mass spectrometry in DBS of VLCADD patients (n = 15) and matched controls. Multivariate analysis of the metabolomic profiles was applied to differentiate subgroups. RESULTS: Concentration of six acylcarnitine species differed significantly between patients and controls. The concentration of C18:2- and C20:0-carnitine, 13,14-dihydroretinol and deoxycytidine monophosphate allowed separation between mild and severe patients. Two patients who could not be prognosticated on early clinical symptoms, were correctly fitted for severity in the score plot based on the untargeted metabolomics. CONCLUSION: Distinctive metabolomic profiles in DBS of newborns with VLCADD may allow phenotypic prognostication. The full potential of this approach as well as the underlying biochemical mechanisms need further investigation.

Newborn blood spot screening for cystic fibrosis with a four-step screening strategy in the Netherlands.

BACKGROUND: Newborn screening for cystic fibrosis (NBSCF) was introduced in the Dutch NBS program in 2011 with a novel strategy. METHODS: Dutch NBSCF consisted of four steps: immuno-reactive trypsin (IRT), Pancreatitis-associated Protein (PAP), DNA analysis by Inno-LiPa (35 mutations), extended gene analysis (EGA) as fourth step and as safety net. Only samples with two CFTR-variants were considered screen-positive, but samples with one disease-causing variant were considered also screen-positive from April 2013. The first 5 years of NBSCF were evaluated during a follow-up ranging from 2 to 6.8 years for sensitivity, specificity, positive predictive value (PPV), ratio of CF/Cystic Fibrosis Screen Positive infants with an Inconclusive Diagnosis (CFSPID) and median age at diagnosis, and were compared to other novel strategies for NBSCF and European Cystic Fibrosis Society (ECFS) Best Practice Standards of Care. RESULTS: NBSCF achieved a sensitivity of 90% (95% CI 82%-94%), specificity of 99.991% (95% CI 99.989%-99.993%), PPV of 63% (95% CI 55%-69%), CF/CFSPID ratio of 4/1, and median age at diagnosis of 22 days, if samples with two variants as well as samples with one disease-causing variant were considered screen-positive. CONCLUSION: The program achieved the goal to minimize the number of false positives and showed a favourable performance but sensitivity and CF/CFSPID ratio did not meet criteria of EFCS Best Standards of Care. Changed cut-off values for PAP and IRT and classification of R117H-7T/9T to non-pathogenic may improve sensitivity to ≥95% and CF/CFSPID ratio to 10/1. PPV is estimated to be around 60%.

Newborn Screening for Lysosomal Storage Diseases: A Concise Review of the Literature on Screening Methods, Therapeutic Possibilities and Regional Programs.

Newborn screening for lysosomal storage diseases (LSDs) is increasingly being considered as an option. The development of analytical screening methods, of second-tier methods, and of therapeutic possibilities, are paving the way for routine screening for LSDs in the coming years. Here, we give a brief description of the current status quo, what screening methods are currently available or are in the pipeline, what is the current status of therapeutic possibilities for LSDs, what LSDs are the most obvious candidates for introduction in screening programs, and what LSDs are already part of regional or national pilot or routine screening programs worldwide.

Perspectives, preferences and needs regarding early prediction of preeclampsia in Dutch pregnant women: a qualitative study.

BACKGROUND: To improve early risk-identification in pregnancy, research on prediction models for common pregnancy complications is ongoing. Therefore, it was the aim of this study to explore pregnant women's perceptions, preferences and needs regarding prediction models for first trimester screening for common pregnancy complications, such as preeclampsia, to support future implementation. METHOD: Ten focus groups (of which five with primiparous and five with multiparous women) were conducted (n = 45). Six focus groups were conducted in urban regions and four in rural regions. All focus group discussions were audio taped and NVIVO was used in order to facilitate the thematic analysis conducted by the researchers. RESULTS: Women in this study had a positive attitude towards first trimester screening for preeclampsia using prediction models. Reassurance when determined as low-risk was a major need for using the test. Self-monitoring, early recognition and intensive monitoring were considered benefits of using prediction models in case of a high-risk. Women acknowledged that high-risk determination could cause (unnecessary) anxiety, but it was expected that personal and professional interventions would level out this anxiety. CONCLUSION: Women in this study had positive attitudes towards preeclampsia screening. Self-monitoring, together with increased alertness of healthcare professionals, would enable them to take active actions to improve pregnancy outcomes. This attitude enhances the opportunities for prevention, early recognition and treatment of preeclampsia and probably other adverse pregnancy outcomes.

Explaining variation in Down's syndrome screening uptake: comparing the Netherlands with England and Denmark using documentary analysis and expert stakeholder interviews.

BACKGROUND: The offer of prenatal Down's syndrome screening is part of routine antenatal care in most of Europe; however screening uptake varies significantly across countries. Although a decision to accept or reject screening is a personal choice, it is unlikely that the widely differing uptake rates across countries can be explained by variation in individual values alone.The aim of this study was to compare Down's syndrome screening policies and programmes in the Netherlands, where uptake is relatively low (<30%) with England and Denmark where uptake is higher (74 and > 90% respectively), in an attempt to explain the observed variation in national uptake rates. METHODS: We used a mixed methods approach with an embedded design: a) documentary analysis and b) expert stakeholder analysis. National central statistical offices and legal documents were studied first to gain insight in demographic characteristics, cultural background, organization and structure of healthcare followed by documentary analysis of primary and secondary sources on relevant documents on DSS policies and programme. To enhance interpretation of these findings we performed in-depth interviews with relevant expert stakeholders. RESULTS: There were many similarities in the demographics, healthcare systems, government abortion legislation and Down's syndrome screening policy across the studied countries. However, the additional cost for Down's syndrome screening over and above standard antenatal care in the Netherlands and an emphasis on the 'right not to know' about screening in this country were identified as potential explanations for the 'low' uptake rates of Down's syndrome screening in the Netherlands. The social context and positive framing of the offer at the service delivery level may play a role in the relatively high uptake rates in Denmark. CONCLUSIONS: This paper makes an important contribution to understanding how macro-level demographic, social and healthcare delivery factors may have an impact on national uptake rates for Down's syndrome screening. It has suggested a number of policy level and system characteristics that may go some way to explaining the relatively low uptake rates of Down's syndrome screening in the Netherlands when compared to England and Denmark.

Comparison of different blood collection, sample matrix, and immunoassay methods in a prenatal screening setting.

We compared how measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic gonadotropin (fß-hCG) in maternal blood are influenced by different methods for blood collection, sample matrix, and immunoassay platform. Serum and dried blood spots (DBS) were obtained by venipuncture and by finger prick of 19 pregnant women. PAPP-A and fß-hCG from serum and from DBS were measured by conventional indirect immunoassay on an AutoDELFIA platform and by antibody microarray. We compared methods based on the recoveries for both markers as well as marker levels correlations across samples. All method comparisons showed high correlations for both marker concentrations. Recovery levels of PAPP-A from DBS were 30% lower, while those of fß-hCG from DBS were 50% higher compared to conventional venipuncture serum. The recoveries were not affected by blood collection or immunoassay method. The high correlation coefficients for both markers indicate that DBS from finger prick can be used reliably in a prenatal screening setting, as a less costly and minimally invasive alternative for venipuncture serum, with great logistical advantages. Additionally, the use of antibody arrays will allow for extending the number of first trimester screening markers on maternal and fetal health.

Positive predictive values for detection of trisomies 21, 18 and 13 and termination of pregnancy rates after referral for advanced maternal age, first trimester combined test or ultrasound abnormalities in a national screening programme (2007-2009).

OBJECTIVE: The objective of this article is to analyse the positive predictive value (PPV) of trisomies 21, 18 and 13 after referral for advanced maternal age (AMA), first trimester combined test or ultrasound findings to suggest improvements for clinical practice. METHODS: Data (48 457 combined tests, 134 000 fetal anomaly scans and 24 379 invasive prenatal tests) were combined to calculate PPV and termination of pregnancy rates. RESULTS: For referral for AMA, the PPV for T21 was 1.0% and 1.8% for amniocentesis and chorionic villus biopsy, respectively; for the combined test at a maternal age ≥36 years, these percentages were 4.9% and 12.5%, respectively and for maternal age <36 years, 4.4% and 8.1%, respectively. For ultrasound findings, the PPV was 5.3% and 14.8%, respectively. Termination of pregnancy rate upon trisomy 21 diagnosis was >90% unless detected after referral for ultrasound findings (71.5-85.9%). About 50% of pregnant women with a high combined test risk chose not to have invasive testing. CONCLUSIONS: Advanced MA is still a large contributor to invasive testing but should be abandoned (low PPV, high fetal loss rate) and be replaced by reimbursable combined test screening for all women. Patient information on second trimester ultrasound screening should indicate that abnormal ultrasound findings are associated with high trisomy rate.

Identification of interleukin-1 beta, but no other inflammatory proteins, as an early onset pre-eclampsia biomarker in first trimester serum by bead-based multiplexed immunoassays.

OBJECTIVE: This study aimed to determine the predictive value of growth factors, cardiovascular, and immunological markers for first trimester identification of early onset pre-eclampsia (PE). METHODS: In a retrospective case-control study, maternal serum samples of 35 early onset PE cases and 35 controls were analysed by multiplexed immunoassays, to determine serum concentrations of 41 proteins whose functionality can be associated with PE pathogenesis. All levels were converted into multiples of the gestation-specific normal median. For prediction modelling, proteins that were found to be significant were combined with previously obtained values of three established PE markers, that is, placental growth factor, placental protein 13, and pregnancy-associated plasma protein A. Prediction modelling was used to determine predicted detection rates for 5% and 10% false-positive rates. RESULTS: Three of the proteins examined in this study, interleukin-1 beta (IL-1ß), fibrinogen, and carcinoembryonic antigen, showed significantly different serum levels at p < 0.05. In prediction modelling, only IL-1ß added predictive value to the three previously established biomarkers, by increasing detection from 38.2% to 44.1% at a 5% false-positive rate. CONCLUSIONS: This study indicates that IL-1ß has potential to improve first trimester prediction of pre-eclampsia. Studies on larger cohorts will be needed to validate these findings.

Comparison of risk calculation approaches in a screening programme for Down syndrome.

In the Netherlands, both the LifeCycle Elipse (LC) and the Astraia software package are used to calculate the risk of having a child with Down syndrome. Therefore, pregnant women can be presented with dissimilar risks. In this study the conformity between these risks before and after harmonization of the screening program and its influence on the performance indicators of the first trimester screening were evaluated. The agreement between combined risks (based on the biochemical parameters PAPP-A and fß-hCG and a nuchal translucency measurement) was expressed as intraclass correlation coefficient (ICC)=0.99. Conformity between combined risks was better after harmonization (Cohen's κ=0.75) than before harmonization (Cohen's κ=0.63). For both risk calculation software packages the area under the ROC-curve was 0.84. The database contained 42 Down syndrome cases; based on the odds of being affected given a positive result (OAPR), LC performed slightly better than Astraia before harmonization (17.9 vs. 21.5, respectively). It has been acknowledged that using different software packages could lead to dissimilar risk calculations. In this study the screening performance indicators of two software packages were quite similar. The agreement of the screening performance after harmonization remains to be seen, but is expected to be even higher.

Integrative data mining to identify novel candidate serum biomarkers for pre-eclampsia screening.

OBJECTIVE: Pre-eclampsia (PE) is a serious complication that affects approximately 2% of pregnant women worldwide. At present, there is no sufficiently reliable test for early detection of PE in a screening setting that would allow timely intervention. To help future experimental identification of serum biomarkers for early onset PE, we applied a data mining approach to create a set of candidate biomarkers. METHODS: We started from the disease etiology, which involves impaired trophoblast invasion into the spiral arteries. On the basis of this, we used a three-stage filtering strategy consisting of selection of tissue-specific genes, textmining for further gene prioritization, and identifying blood-detectable markers. RESULTS: This approach resulted in 38 candidate biomarkers. These include the best three first-trimester serum biomarkers for PE found to date LGALS13 (placental protein 13, PP13), PAPPA (pregnancy-associated plasma protein-A, PAPP-A), and PGF (placental growth factor, PlGF), as well as five proteins previously identified as biomarker after the first-trimester or disease onset. This substantiates the effectiveness of our approach and provides an important indication that the list will contain several new biomarkers for PE. CONCLUSIONS: We anticipate this list can serve in prioritization of future experimental studies on serum biomarkers for early onset PE.

Trends in the utilization of invasive prenatal diagnosis in The Netherlands during 2000-2009.

OBJECTIVE: To analyze trends in the number and type of invasive procedure, reasons for referral, maternal age and chromosomal abnormalities over a 10-year period and correlate the trends to changes in the national prenatal screening policy. METHODS: Data from 10 706 invasive prenatal procedures yielding a full karyotype, performed between 2000 and 2009 were extracted from the cytogenetic database in the central region of The Netherlands. Trends were analyzed. RESULTS: Over a 10-year period, the number of invasive procedures halved and the percentage of chromosomal abnormalities detected, increased from 5.5 to 9.4%. After 2007, however, 5.7% of karyotypes in women over 36 years were found to be abnormal, versus 18.1% in women below 36 years. In 2009, 71.5% of women over 36 are still referred for invasive prenatal diagnosis on the indication advanced maternal age. CONCLUSIONS: Changes in prenatal screening policy significantly increased referral after screening and improved the efficacy of invasive prenatal diagnosis. We show the continuing effect of the different policies applied in the past to women below and above the age of 36. To further improve efficacy of invasive prenatal diagnosis, first trimester combination screening should be actively offered to women of all ages.

Performance of free beta-human chorionic gonadotrophin (free beta-hCG) and pregnancy associated plasma protein-A (PAPP-A) analysis between Delfia Xpress and AutoDelfia systems in The Netherlands.

BACKGROUND: The VU University Medical Center (VUmc) was the first hospital in the Netherlands to introduce the Delfia Xpress for the analysis of free beta-human chorionic gonadotrophin (beta-hCG) and pregnancy associated plasma protein-A (PAPP-A) in the first trimester screening program for Down syndrome. Since then, others have implemented this system. In this study, we tested the equality of measurements for free beta-hCG and PAPP-A between Delfia Xpress systems and one AutoDelfia system. METHODS: A total of 40 serum samples were aliquoted and stored at -20 degrees C. Samples were analyzed by six Delfia Xpress systems and one AutoDelfia system over a time period of 2 years. RESULTS: The relationships between free beta-hCG and PAPP-A were excellent for the different Delfia Xpress systems (r>0.99, p<0.0001). For PAPP-A, the agreement between the main system at VUmc and five other systems was linear with slopes between 0.99 and 1.06. Similarly, agreement for free beta-hCG was linear with slopes between 0.99 and 1.09. Likewise, agreement for PAPP-A and free beta-hCG was excellent for the AutoDelfia vs. the main Delfia Xpress at the VUmc (r>0.99, p<0.0001). For both PAPP-A and free beta-hCG, the relationships were linear with slopes of 1.08 and 1.07. CONCLUSIONS: We demonstrate an excellent agreement for the analysis of PAPP-A and free beta-hCG between Delfia Xpress systems and one AutoDelfia system.

Costs and effects of prenatal screening methods for Down syndrome and neural tube defects.

OBJECTIVES: To evaluate prenatal screening methods for Down syndrome and neural tube defects (NTD) with regard to costs per detected case and the number of screening-related miscarriages. METHODS: The screening methods compared were risk assessment tests, i.e. serum tests and nuchal translucency measurement (NT), and invasive testing through chorionic villus sampling (CVS) or amniocentesis. Costs, the number of cases detected and screening-related miscarriages were calculated using a decision tree model. RESULTS: The costs per detected case of Down syndrome ranged from EUR 98,000 for the first-trimester (serum) double test to EUR 191,000 for invasive testing. If NTD detection was included, the (serum) triple test had the lowest costs, EUR 73,000, per detected case of Down syndrome or NTD. The number of screening-related miscarriages due to invasive diagnostic tests varied from 13 per 100,000 women for the (serum) first- and second-trimester combined test to 914 per 100,000 women for invasive testing. CONCLUSIONS: Considering screening for both Down syndrome and NTD favors the triple test in terms of costs per detected case. Compared to invasive testing, risk assessment tests in general substantially lower screening-related miscarriages, which raises the question of whether invasive testing should still be offered in a screening program for Down syndrome.