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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 1.14 μmol/L ± 0.45 standard deviations. The average creatine level was 408 μmol/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.

Objectives To determine whether estimation of gestational age (GA) in the context of first-trimester Down syndrome screening is standardized in the Netherlands. Methods This was a retrospective study, carried out between January 2005 and December 2006, of women who underwent first-trimester Down syndrome screening (n = 40 730) based on GA, maternal serum analysis and nuchal translucency (NT) measurement. Date of the last menstrual period (LMP), dating scan information including measurement of crown–rump length (CRL), NT thickness and name of the sonographer were recorded for all pregnancies. The accuracy of estimation of GA was evaluated by comparing the GA based on the LMP with that estimated from the CRL, using relevant subsets of the database. A survey of 104 sonographers was performed to further investigate the findings of the preceding analysis. Results In 44% of all first-trimester combined tests the estimation of GA was based on the dating scan; the method of determination of GA was unknown in 23%. In 15% of all cases a dating scan was recorded but was not used to provide the estimation of GA at blood sampling. Detailed analysis showed that a consistent methodology for the estimation of GA from CRL was not maintained within hospitals and obstetric practices. For a single CRL, the reported GA differed by up to 10 days. Finally, it was demonstrated that individual sonographers reported different GAs for a given CRL. Conclusions Currently, estimation of GA in the first trimester in the Netherlands is not standardized. To improve the performance of prenatal screening for Down syndrome, estimation of GA should be based on ultrasound examination, with one nationally accepted CRL curve. Copyright © 2008 ISUOG. Published by John Wiley & Sons, Ltd.

Background: Because of lack of worldwide standardization of influenza virus surveillance, comparison between countries of impact of a pandemic is challenging. For that, other approaches to allow internationally comparative serosurveys are welcome. Objectives: Here we explore the use of neonatal screening dried blood spots to monitor the trends of the 2009 influenza A (H1N1) pdm virus by the use of a protein microarray. Study design: We contacted colleagues from neonatal screening laboratories and asked for their willingness to participate in a study by testing anonymized neonatal screening bloodspots collected during the course of the pandemic. In total, 7749 dried blood spots from 13 countries in 5 continents where analyzed by using a protein microarray containing HA1 recombinant proteins derived from pandemic influenza A (H1N1) 2009 as well as seasonal influenza viruses. Results: Results confirm the early start of the pandemic with extensive circulation in the US and Canada, when circulation of the new virus was limited in other parts of the world. The data collected from sites in Mexico suggested limited circulation of the virus during the early pandemic phase in this country. In contrast and to our surprise, an increase in seroprevalence early in 2009 was noted in the dataset from Argentina, suggestive of much more widespread circulation of the novel virus in this country than in Mexico. Conclusions: We conclude that this uniform serological testing of samples from a highly standardized screening system offers an interesting opportunity for monitoring population level attack rates of widespread diseases outbreaks and pandemics. (C) 2014 The Authors. Published by Elsevier B.V.

OBJECTIVES: This is the first report on the results of a first-trimester combined-test screening programme in the Netherlands in a multi-centre routine clinical setting. METHODS: Between July 2002 and May 2004, blood samples were taken from subjects in 44 centres in the Netherlands and sent to our laboratory to assay for maternal serum concentrations of fbeta-hCG and PAPP-A. Fetal nuchal translucency (NT) was measured in the participating centres at a gestational age (GA) of 10-14 weeks. Results of those pregnancies for which a combined biochemical and NT risk was calculated were included in the epidemiological analysis of this study. RESULTS: A total of 4033 singleton pregnancies were included in the analysis. The median maternal age of the analysed group was 36.5 years. The distribution of GA was biphasic, with median GA of 10.3 and 12.1 weeks, respectively. The detection rate using the combined ultrasound and serum screening at a cut-off level of 1 in 250 was 71% (15/21), with a screen-positive rate of 4.7%. CONCLUSION: The results of this study show that the first-trimester combined test is suitable as a prenatal screening test in a multi-centre routine clinical setting in the Netherlands. Strict performance evaluation should identify weaknesses in the organisation that impair the performance of the test. Here, the performance of NT was especially identified as a candidate for improvement.

Objective To develop a standard preparation for measurement of phenylalanine in blood spots in phenylketonuria against which manufacturers of kits for phenylalanine determination could calibrate their own blood spot calibrators. Methods As most of the variation of phenylalanine concentration in dried blood spots is due to the way the blood spot calibrators are prepared, a minimum set of prerequisites for the preparation of blood spot calibrators was established by a group of scientific experts and manufacturers. A “European working standard for phenylalanine in blood spots” (EWS-Phe-01) was prepared and distributed to manufacturers taking part in this project. Results The control of the homogeneity, linearity, and stability of the EWS-Phe-01 batch gave results within expected ranges. The preliminary results of the recalibration of calibrators for phenylalanine against EWS-Phe-01 by the participating manufacturers were promising; the differences between measurements were much decreased. Conclusion Manufacturers have agreed to use EWS-Phe-01, and since November 1996 EWS-Phe calibrators have been routinely available from these companies. This pilot study may act as a model for minimising discrepancies found in other screening tests.

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.

Summary From the results of this study the conclusion can be drawn that radioimmunoassay (RIA) of diethylstilbestrol (DES) in crude urinary extracts may lead to highly unreliable results. Simple paper‐chromatographic purification, prior to radioimmunoassay, eliminates interfering substances responsible for ‘false DES’ values. Such a purification step is mandatory, certainly when the radioimmunoassay is used for statutory control purposes.

beta-Endorphin (beta-LPH61-91) is a well known endogenous opioid ligand. It and related peptides have recently been implicated in the control of adaptive behaviour. Smaller beta-endorphin fragments appeared to be more active moieties than the parent molecule in a number of behavioural situations. Their effects seemed to occur independently of interaction with opiate receptor sites in the brain. Moreover, elimination of the opiate-like properties of gamma-endorphin (beta-LPH61-77) by removing the N-terminal amino acid yielded des-tyrosine-gamma-endorphin (beta-LPH62-77, dT gamma E) which had greater behavioural activity than gamma-endorphin. The CNS effects of dT gamma E resembled those of neuroleptic drugs in several test systems. alpha-Endorphin (beta-LPH61-76) exerted effects opposite to those of dT gamma E and in some aspects its activity was comparable to that of psychostimulant drugs. This opposition of effects suggests that a balance between gamma- and alpha-type endorphins is involved in the control of brain function. We report here that either gamma-endorphin and dT gamma E or alpha-endorphin and des-tyrosine-alpha-endorphin (beta-LPH62-76, DT alpha E) can be formed preferentially from beta-endorphin by enzymes associated with an enriched synaptosomal plasma membrane fraction from brain. It is suggested that these enzymes have a role in brain homeostatic mechanisms by regulating the generation of these substances.

Summary: Six commercially available radioimmunoassay (RIA) kits for human follitropin (follicle-stimulating hormone; hFSH) were compared with respect to precision, "accuracy", specificity and performance. The intra- and interassay coefficients of Variation ranged from 4.7 to 8.3% and from 6.5 to 13.3%, respectively. The recovery of added follitropin ranged from 88 to 176%. The most important conclusion of this study is that an accurate calibration of the kit Standard by the manufacturer is essential for minimizing the Variation between kits. « Vergleich von sechs Radioimmunoassay-Bestecks zur Bestimmung von menschlichem Follitropin im Serum Zusammenfassung: Sechs kommerziell erhaltliche Radioimmunassay-Bestecks zur Bestimmung menschlichen Follitropin wurden hinsichtlich Prazision, Richtigkeit, Spezifitat und Durchfuhrung verglichen. Die Variationskoeffizienten betrugen in der Serie 4,7 bis 8,3%, von Tag zu Tag 6,5 bis 13,3%. Die Wiederfindung zugesetzten Foljitropins reichte von 88 bis 176%. Die wichtigste Schlusfolgerung aus dieser Untersuchung ist, das eine richtige Kalibrierung des Standards im Testbesteck durch den Hersteller fur die Verminderung der Variation der mit den Bestecks erzielten Ergebnisse von ausschlaggebender Bedeutung ist.

Door het toevoegen van kleurstoffen aan reagentia van RIA systemen is controle op het pipetteren en eventueel morsen (van radioactiviteit) eenvoudig. Een twaalftal in de voedingsmiddelen industrie veel gebruikte kleurstoffen werd in acht in eigen beheer ontwikkelde RIA systemen getest met betrekking tot verstoring van initiele en aspecifieke binding. Vrijwel alle kleurstoffen zijn bruikbaar in een concentratie van 50 mg/l incubatievolume.

Neonatal screening for congenital disorders like phenylketonuria (PKU), congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) is generally performed in dried blood spots on filter paper. The analytes of interest for testing for PKU, CH and CAH are phenylalanine, thyrotropin (TSH) and 17alpha-hydroxyprogesterone (17OHP), respectively. The International Society for Neonatal Screening (ISNS) decided to prepare a combined reference preparation for the three analytes on filter paper SchleicherSchuell #903, Whatman BFC180 and Toyo Roshi 545. This 'First ISNS Reference Preparation for Neonatal Screening for TSH, phenylalanine and 17OHP in blood spots' (1st ISNS-RPNS) has been prepared by the RIVM (Bilthoven).The number of filter paper cards prepared, each with two sets of six blood spot calibrators, was 480, 42 and 69 for SchleicherSchuell #903, Whatman BFC180 and Toyo Roshi 545, respectively. The volume of blood dispensed was 50 microl. The range of concentrations for TSH was 1-121 mIU/L blood, for phenylalanine 65-865 micromol/L blood and for 17OHP 2.2-302 nmol/L blood.The linearity of the blood spot calibrators and the homogeneity of the batch (only tested for SchleicherSchuell) were good. The differences between the three filter papers were small: i.e. the potency of the ISNS-RPNS on Whatman and Toyo Roshi in terms of SchleicherSchuell was between 0.98 and 1.09 for the three analytes.The 1st ISNS-RPNS for TSH, phenylalanine and 17OHP can be said to be suitable as formal reference preparation and as a source for (re)calibrating kit calibrators.