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1.  Robustness of genome-wide scanning using archived dried blood spot samples as a DNA source 
BMC Genetics  2011;12:58.
The search to identify disease-susceptible genes requires access to biological material from numerous well-characterized subjects. Archived residual dried blood spot (DBS) samples, also known as Guthrie cards, from national newborn screening programs may provide a DNA source for entire populations. Combined with clinical information from medical registries, DBS samples could provide a rich source for productive research. However, the amounts of DNA which can be extracted from these precious samples are minute and may be prohibitive for numerous genotypings. Previously, we demonstrated that DBS DNA can be whole-genome amplified and used for reliable genetic analysis on different platforms, including genome-wide scanning arrays. However, it remains unclear whether this approach is workable on a large sample scale. We examined the robustness of using DBS samples for whole-genome amplification following genome-wide scanning, using arrays from Illumina and Affymetrix.
This study is based on 4,641 DBS samples from the Danish Newborn Screening Biobank, extracted for three separate genome-wide association studies. The amount of amplified DNA was significantly (P < 0.05) affected by the year of storage and storage conditions. Nine (0.2%) DBS samples failed whole-genome amplification. A total of 4,586 (98.8%) samples met our criterion of success of a genetic call-rate above 97%. The three studies used different arrays, with mean genotyping call-rates of 99.385% (Illumina Infinium Human610-Quad), 99.722% (Illumina Infinium HD HumanOmni1-Quad), and 99.206% (Affymetrix Axiom Genome-Wide CEU). We observed a concordance rate of 99.997% in the 38 methodological replications, and 99.999% in the 27 technical replications. Handling variables such as time of storage, storage conditions and type of filter paper were shown too significantly (P < 0.05) affect the genotype call-rates in some of the arrays, although the effect was minimal.
Our study indicates that archived DBS samples from the Danish Newborn Screening Biobank represent a reliable resource of DNA for whole-genome amplification and subsequent genome-wide association studies. With call-rates equivalent to high quality DNA samples, our results point to new opportunities for using the neonatal biobanks available worldwide in the hunt for genetic components of disease.
PMCID: PMC3142526  PMID: 21726430
2.  Evaluation of Sex-Specific Gene Expression in Archived Dried Blood Spots (DBS) 
Screening newborns for treatable serious conditions is mandated in all US states and many other countries. After screening, Guthrie cards with residual blood (whole spots or portions of spots) are typically stored at ambient temperature in many facilities. The potential of archived dried blood spots (DBS) for at-birth molecular studies in epidemiological and clinical research is substantial. However, it is also challenging as analytes from DBS may be degraded due to preparation and storage conditions. We previously reported an improved assay for obtaining global RNA gene expression from blood spots. Here, we evaluated sex-specific gene expression and its preservation in DBS using oligonucleotide microarray technology. We found X inactivation-specific transcript (XIST), lysine-specific demethylase 5D (KDM5D) (also known as selected cDNA on Y, homolog of mouse (SMCY)), uncharacterized LOC729444 (LOC729444), and testis-specific transcript, Y-linked 21 (TTTY21) to be differentially-expressed by sex of the newborn. Our finding that trait-specific RNA gene expression is preserved in unfrozen DBS, demonstrates the technical feasibility of performing molecular genetic profiling using such samples. With millions of DBS potentially available for research, we see new opportunities in using newborn molecular gene expression to better understand molecular pathogenesis of perinatal diseases.
PMCID: PMC3431816  PMID: 22949818
archived dried blood spots (DBS); sex-specific; gene expression; molecular genetic profiling; microarray
3.  Newborn Screening and the Obstetrician 
Obstetrics and gynecology  2012;120(4):908-917.
Newborn screening is the largest genetic screening program in the United States, with approximately four million infants screened yearly. It has been available and in continuous development for over 50 years. Each state manages, funds, and maintains its own individual program, which encompasses newborn screening as well as the diagnosis and coordination of care for affected infants and children. The ideal disorder for screening is one in which newborn intervention prevents later disabilities or death for infants who may appear normal at birth. There are 31 core conditions that are currently recommended for incorporation into state screening programs. To obtain a sample, several drops of blood are collected from the newborn’s heel and applied to filter paper. Although testing for core disorders is fairly standardized, more extensive screening varies by state and the rigorous evaluation of new disorders for inclusion in state screening panels is ongoing. As genomic medicine becomes more accessible, screening newborns for chronic diseases that may affect their long-term health will need to be addressed, as well the use of the residual blood spots for research. Obstetric providers should, at some time during pregnancy, review the basic process of newborn screening with parents to prepare them for this testing in the neonatal period. This information can be reviewed as it best suits incorporation in an individual’s practice; verbal discussion and the distribution of written materials with resources for further information is encouraged.
PMCID: PMC3459237  PMID: 22996108
4.  High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening 
Clinical chemistry  2013;59(3):502-511.
There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS).
The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre–HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard–specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode.
Analysis of blood spots from 58 random newborns and lysosomal storage disease–affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories.
HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.
PMCID: PMC4737435  PMID: 23315484
5.  State Laws Regarding the Retention and Use of Residual Newborn Screening Blood Samples 
Pediatrics  2011;127(4):703-712.
After newborn screening has been completed, many states retain residual newborn screening dried blood samples for various purposes, including program evaluation, quality assurance, and biomedical research. The extent to which states possess legal authority to retain residual dried blood samples (DBS) and use them for purposes unrelated to newborn screening is unclear.
The purpose of this study was to evaluate state laws regarding the retention and use of DBS.
State statutes and regulations related to newborn screening of all 50 states plus the District of Columbia were accessed online between November 2008 and December 2009 and reviewed by 2 independent reviewers to determine the extent to which the retention and use of DBS were addressed.
The retention or use of DBS has not been addressed in 18 states. In 4 states, DBS becomes state property. Eight states require that parents be provided information regarding the retention of DBS. Parents in 5 states may request the destruction of their child's residual sample. Parental consent is required under certain circumstances to release DBS for research in 6 states. One state prohibits DBS from being used for research purposes.
States have wide variability in their policies regarding the retention and use of DBS. Many states have not addressed key issues, and some states that retain DBS may be acting outside the scope of their legal authority. The lack of transparency on the part of states in retaining DBS may undermine public trust in state newborn screening programs and the research enterprise.
PMCID: PMC3065077  PMID: 21444595
newborn screening; dried blood samples; research; ethics; law; consent; opt-out
6.  Evaluation of Glucose-6-Phosphate Dehydrogenase stability in stored blood samples  
EXCLI Journal  2016;15:155-162.
Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the commonest cause of neonatal jaundice in Malaysia. Recently, OSMMR2000-D G6PD Assay Kit has been introduced to quantitate the level of G6PD activity in newborns delivered in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). As duration of sample storage prior to analysis is one of the matters of concern, this study was conducted to identify the stability of G6PD enzyme during storage. A total of 188 cord blood samples from normal term newborns delivered at UKMMC were selected for this study. The cord bloods samples were collected in ethylene-diamine-tetra-acetic acid (EDTA) tubes and refrigerated at 2-8 °C. In addition, 32 out of 188 cord blood samples were spotted on chromatography paper, air-dried and stored at room temperature. G6PD enzyme activities were measured daily for 7 days using the OSMMR2000-D G6PD Assay Kit on both the EDTA blood and dried blood samples. The mean value for G6PD activity was compared between days of analysis using Student Paired T-Test. In this study, 172 out of 188 cord blood samples showed normal enzyme levels while 16 had levels corresponding to severe enzyme deficiency. The daily mean G6PD activity for EDTA blood samples of newborns with normal G6PD activity showed a significant drop on the fourth day of storage (p < 0.005) while for samples with severely deficient G6PD activity, significant drop was seen on third day of storage (p = 0.002). Analysis of dried cord blood showed a significant reduction in enzyme activity as early as the second day of storage (p = 0.001). It was also noted that mean G6PD activity for spotted blood samples were lower compared to those in EDTA tubes for all days (p = 0.001). Thus, EDTA blood samples stored at 2-8 °C appeared to have better stability in terms of their G6PD enzyme level as compared to dried blood samples on filter paper, giving a storage time of up to 3 days.
PMCID: PMC4834747  PMID: 27103895
G6PD stability; G6PD enzyme assay; OSMMR2000-D G6PD Assay Kit; EDTA; dried blood spot
7.  Use of Tandem Mass Spectrometry for Newborn Screening of 6 Lysosomal Storage Disorders in a Korean Population 
We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome.
We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods.
The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns.
The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.
PMCID: PMC3190003  PMID: 22016678
Lysosomal storage disorders; Multiplex enzyme assay; Tandem mass spectrometry; Newborn screening
8.  Public banking of umbilical cord blood or storage in a private bank: testing social and ethical policy in northeastern Italy 
In northeastern Italy, according to Italian legislation, authorized public facilities can accept the donation and preservation of cord blood stem cells (CB-SC). Attitudes and knowledge in pregnant women differs between the local and immigrant (non-European Union [EU]) population. In this study we assessed the choices that pregnant women have with respect to the public and private harvesting system and the main reasons driving their decisions. We examined the ethnic origin of the families and compared tests for syphilis screening and leukocyte (WBC) counts in the CB-SC bags that are required for validation of the collection.
Out of a population of 3450 pregnant patients at the Institute for Maternal and Child Health of Trieste, northeast Italy, 772 women agreed to cord blood harvesting and the associated lab tests. Of these, 221 women (28.6%) were from immigrant families of non-EU countries. Their ethnic affiliation was recorded, and tests were performed for syphilis screening and for nucleated red blood cell (NRBC) interference with the WBC count in CB-SC bags to assess cellularity and to determine if storage was appropriate.
Of the 772 pregnant women, 648 (84.0%) accessed the public collection system, which is free of charge, and 124 (15.0%) accessed the private fee-based system. One woman from the non-EU group opted for the private fee-based system. Of the 3450 pregnant women screened for syphilis at the Institute for Maternal and Child Health, the Treponema pallidum hemagglutination (TPHA) and Venereal Disease Research Laboratory (VDRL) tests were the main tests performed (66.0% of total cases) because many gynecologists in the public harvesting system apply the Italian regulations of the 1988 Decree, while the private system requires tests on syphilis and leaves the option to the lab physicians to select the best determination method. We found that the chemiluminescence method was more specific (97.0%) than the TPHA (83.0%) and nontreponemal rapid plasma reagin VDRL (75.0%) tests (P < 0.05, χ2 test). The specificity link between the two automatic methods versus microscopes for WBC dosing and NRBC interference was r2 = 0.08 (ADVIA 120) and r2 = 0.94 (XE-2100). The public system does not include human T-cell lymphotropic virus testing; this is reserved for the population from endemic zones.
In northeastern Italy current legislation prevents the establishment of private fee-based banks for storage of CB-SC. The cryopreservation, for future autologous personal or family use, is possible only by sending to foreign private banks, with a further fee of €300. These regulations confirm that Italian legislation tries to increase the anonymous allogenic donations and the number of CB-CS bags stored in the free-cost public system, that are available to anyone with therapeutic needs. Private banking is used almost exclusively by the wealthier local population. In the public system, many physicians continue to use older Italian laws regarding syphilis diagnosis, and NRBC interference on WBC count may have an impact on cord blood harvesting. Our findings suggest that in the EU there is no consensus policy on donor management. The value of storage for potential use within the family is useful only with collaboration between the public and the private systems.
PMCID: PMC3628527  PMID: 23610532
cord blood collection; public system; private system; pregnant women’s choice
9.  Sickle cell disease in areas of immigration of high-risk populations: a low cost and reproducible method of screening in northern Italy 
Blood Transfusion  2014;12(3):346-351.
From 2005 to 2010, we observed a 10-fold increase of newly diagnosed sickle cell disease in children in the province of Modena (northern Italy). The median age at diagnosis was 24 months. Since these children are too old for optimal disease management, earlier detection of the disease is needed for prophylaxis and comprehensive care before the occurrence of clinical manifestations.
Materials and methods
In each Maternity Unit of the province of Modena, blood samples are collected daily for assessment of haemolytic disease of the newborn. We designed a selective, low-cost haemoglobin screening for sickle cell disease in high-risk immigrants. We enrolled 469 mothers from sub-Saharan countries and their neonates for a primary screening of peripheral blood haemoglobin variants using high-performance liquid chromatography.
Of the 469 women approached, 330 (70.36%) agreed to undergo the test. Ninety-two (27.88%) were carriers of variant haemoglobin, 48 newborns (51%) of these carriers had the carrier trait and 9 (9.6%) were affected (haemoglobin SC compound heterozigote - HbSC, haemoglobin S homozygote - HbSS).
These results support the feasibility and usefulness of a selective screening for the detection of haemoglobin variants in high-risk subjects in an area in which sickle cells disease is not endogenous. We achieved the goal of detecting subjects with carrier trait/disease in order to implement preventive measures that reduce the clinical manifestations of sickle cell disease. We are, however, aware that it will be necessary to extend this screening to the overall population in the near future.
PMCID: PMC4111816  PMID: 24887233
haemoglobinopathies screening; sickle cell disease and trait; immigrants
10.  Meeting Report: The Use of Newborn Blood Spots in Environmental Research: Opportunities and Challenges 
Environmental Health Perspectives  2007;115(12):1767-1779.
Dried blood spots (DBS) are routinely collected from newborns in the United States using a heel stick. The DBS are screened for inborn errors of metabolism and other disorders. More states are keeping residual spots and making them available for research purposes. DNA extraction from the DBS has been widely applied; however, the development of methods to measure a range of environmental toxicants in DBS has been a more recent goal for laboratory scientists and epidemiologists.
The purpose of the meeting was to examine the utility of DBS to measure environmental exposures. Speakers and discussants were invited to present data and discuss approaches to measure a range of analytes using DBS.
This meeting was held on 20 February 2007 at the University of North Carolina at Chapel Hill. The audience consisted of epidemiologists, chemists, and staff from state public health programs, the Centers for Disease Control and Prevention, and the National Institutes of Health. The meeting included presentations on measurement of flame-retarding chemicals and pesticides, metals, perchlorate, infectious agents, markers of immune status, and protein adducts. Analytical methods included mass spectrometry, atomic absorption, molecular methods, and microfluidic techniques. Significant progress was reported, but important challenges remain. Concerns including storage conditions, sample volume, contamination, and normalization require additional systematic evaluation. In addition, DBS storage and access policies require coordination.
DBS remain a highly valuable resource for clinical, epidemiologic, and toxicologic investigation. The use of DBS to measure environmental exposures shows promise but additional work is necessary before more widespread use is warranted.
PMCID: PMC2137126  PMID: 18087597
environmental monitoring; environmental pollutants; infant; neonatal screening; newborn; specimen handling
11.  Fragile X protein in newborn dried blood spots 
BMC Medical Genetics  2014;15:119.
The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. We previously characterized 215 dried blood spots (DBS) representing different FMR1 genotypes and ages with a Luminex-based immunoassay (qFMRP). We found variable FMRP levels in the normal samples and identified affected males by the drastic reduction of FMRP.
Here, to establish the variability of expression of FMRP in a larger random population we quantified FMRP in 2,000 anonymous fresh newborn DBS. We also evaluated the effect of long term storage on qFMRP by retrospectively assaying 74 aged newborn DBS that had been stored for 7-84 months that included normal and full mutation individuals. These analyses were performed on 3 mm DBS disks. To identify the alleles associated with the lowest FMRP levels in the fresh DBS, we analyzed the DNA in the samples that were more than two standard deviations below the mean.
Analysis of the fresh newborn DBS revealed a broad distribution of FMRP with a mean approximately 7-fold higher than that we previously reported for fresh DBS in normal adults and no samples whose FMRP level indicated FXS. DNA analysis of the lowest FMRP DBS showed that this was the low extreme of the normal range and included a female carrying a 165 CGG repeat premutation. In the retrospective study of aged newborn DBS, the FMRP mean of the normal samples was less than 30% of the mean of the fresh DBS. Despite the degraded signal from these aged DBS, qFMRP identified the FXS individuals.
The assay showed that newborn DBS contain high levels of FMRP that will allow identification of males and potentially females, affected by FXS. The assay is also an effective screening tool for aged DBS stored for up to four years.
PMCID: PMC4412103  PMID: 25348928
Fragile X syndrome; FMR1; FMRP; Dried blood spots; DBS; Capture immune assay; CGG repeats; Luminex; Newborn screening
12.  Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening 
The interest in screening lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within four hours including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, multidimensional UHPLC-tandem mass spectrometer. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day. Due to high grade of automation and simplified sample preparation, this assay could be used for urgent clinical diagnostics needed for suspected patients admitted to a hospital, as well as for routine newborn and high-risk population screening.
PMCID: PMC4539023  PMID: 23122395
short-incubation; lysosomal storage disorders; multiplex assay; tandem mass spectrometry; high-throughput; newborn screening; TurboFlow chromatography
13.  Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders 
Tandem mass spectrometry is currently used in newborn screening programmes to quantify the level of amino acids and acylcarnitines in dried blood spots for detection of metabolites associated with treatable diseases. We have developed assays for lysosomal enzymes in re-hydrated dried blood spots in which a set of substrates is added and the set of corresponding enzymatic products are quantified using tandem mass spectrometry with the aid of mass-differentiated internal standards. We have developed a multiplex assay of the set of enzymes that, when deficient, cause the lysosomal storage disorders Fabry, Gaucher, Hurler, Krabbe, Niemann–Pick A/B and Pompe diseases. These diseases were selected because treatments are now available or expected to emerge shortly. The discovery that acarbose is a selective inhibitor of maltase glucoamylase allows the Pompe disease enzyme, acid α-glucosidase, to be selectively assayed in white blood cells and dried blood spots. When tested with dried blood spots from 40 unaffected individuals and 10–12 individuals with the lysosomal storage disorder, the tandem mass spectrometry assay led to the correct identification of the affected individuals with 100% sensitivity. Many of the reagents needed for the new assays are commercially available, and those that are not are being prepared under Good Manufacturing Procedures for approval by the FDA. Our newborn screening assay for Krabbe disease is currently being put in place at the Wadsworth Center in New York State for the analysis of ~1000 dried blood spots per day.
Summary We have developed tandem mass spectrometry for the direct assay of lysosomal enzymes in rehydrated dried blood spots that can be implemented for newborn screening of lysosomal storage disorders. Several enzymes can be analysed by a single method (multiplex analysis) and in a high-throughput manner appropriate for newborn screening laboratories.
PMCID: PMC2488386  PMID: 16763908
14.  AB108. The appliance of Bio-Plex immunoassay using dried blood spots for mucopolysaccharidosis IVA newborn screening in Taiwan—a pilot study 
Annals of Translational Medicine  2015;3(Suppl 2):AB108.
Mucopolysaccharidosis (MPS) IVA is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS) resulting in excessive lysosomal storage of keratan sulfate. This excessive storage causes a systemic skeletal dysplasia, short stature, and joint abnormalities. Treatments for MPS IVA are available. Better outcomes are associated with early treatment, which highlights a need for newborn screening for MPS IVA.
We have conducted a newborn screening pilot program for MPS IVA since December 1, 2013. Screening involved measuring the quantity of GALNS in dried blood spots on filter paper (DBFP) from newborns using a Bio-Plex immunoassay. The amounts of fluorescence sorting detected by YAG laser with wavelengths of 532 (exciting) and 580 nm (emission) is proportional to the quantity of GALNS protein.
More than 5,657 neonates have been analyzed, in those, 132 newborns had GALNS quantification less than the cut-off value (48.64 ρg/mL) at the first screening test. Most of them (n=124) were exclusive and only eight had been recalled for a second DBFP collection and GALNS quantity rechecked. The reference values were 48.64-552.4 ρg/mL. For the confirmed MPS IV patients without enzyme replacement therapy (n=11), the GALNS quantities were far less than 5% of the normal population, and ranged from 0.00 to 4.02 ρg/mL. The GALNS quantities of the carriers (n=2) were significantly reduced comparing with those of the normal values.
The Bio-Plex immunoassay has the potential to be adopted for newborn screening of MPS IVA. This method is reliable, sensitive, validated, simple, and cost-effective in measuring GALNS enzyme in DBFP.
PMCID: PMC4563456
Bio-Plex immunoassay; keratan sulfate; Morquio A syndrome; N-acetylgalactosamine-6-sulfatase (GALNS); newborn screen
15.  AB093. A case of exogenous C5-acylcarnitine giving rise to a false positive result in newborn screening (NBS) 
Annals of Translational Medicine  2015;3(Suppl 2):AB093.
Background and objective
NBS Screening by MS/MS is considered an effective screening test. However, the technique cannot distinguish between isobaric compounds, thus contributing to some false positive results. One such compound is C5-acylcarnitine in the in the identification of isovaleric acidemia (IVA) in the MS/MS profile. To report and contrast the findings of two newborns with C5-acylcarnitine elevations in newborn screening (NBS).
Blood collected on Guthrie card from newborns between 24-72 hours of life is analyzed by MS/MS. C5-acylcarnitine and its related ratios are measured in DBS sample to identify at risk newborn.
Newborn A: DBS sample C5: 7.96 µmol/L (normal <0.50), C5/C0: 0.99 (normal <0.025), C5/C3: 16.1 (normal <0.40); Plasma acylcarnitines profile: C5: 9.42 µmol/L (normal 0.06-0.29). Urine organic acid profiles showed marked elevations of isovalerylglycine (IVG), ketone bodies and lactate. This profile is consistent with a patient presenting with a diagnosis of IVA. Newborn B: DBS sample C5: 0.84 µmol/L (normal <0.50), C5/C0: 0.041 (normal <0.025), C5/C3: 0.46 (normal <0.40); Despite the abnormal plasma acylcarnitines profile (C5: 4.38 µmol/L, normal 0.06-0.29), the urine acylglycine profile was normal [IVG: 1.02 mg/g Cr (normal 0.3-14.3 mg/g); 2-MBG: 0.16 mg/g Cr (normal: 0.3-7.5 mg/g)]. A 2nd plasma acylcarnitine showed a lower C5 level (1.49 µmol/L) and a repeat urine organic acid profile was normal; no IVG and 2-MBG detected. Mother’s (Newborn B) plasma acylcarnitines and urine organic acid profiles were normal, ruling out a possible maternal condition. Moreover, it was confirmed that mother and newborn were not on any antibiotics or steroids, which have been previously reported as the causal agents of falsely elevated C5-acylcarnitine. Further investigation revealed mom was using Mustela Nursing Comfort Balm which contained neopentanoate, a compound demonstrated by Boemer et al. [2014] as the causal agent for the false elevation of C5-acylcarnitine in NBS. The elevated C5 levels in the newborn’s plasma samples appear to correspond to the timing of the feed with the blood draw (i.e., the high C5 plasma acylcarnitine result (4.38 µmol/L) corresponds to a blood sample taken within minutes of a feed and the second sample was collected several hours (>2 hours) after the feed (C5: 1.49 µmol/L). Withdrawal of Comfort Balm use eliminated the biochemical derangements in Newborn B and he is clinically well at 10 months.
Neopentanoate in the form of an emollient can cause a C5 false positive result in NBS.
PMCID: PMC4563512
Newborn screening (NBS); C5-acylcarnitine; isovaleric academia (IVA)
16.  Efficiency of a solid-phase chemiluminescence immunoassay for detection of antinuclear and cytoplasmic autoantibodies compared with gold standard immunoprecipitation 
Auto-Immunity Highlights  2014;5(2):47-54.
The aim of this study was to compare the degree of agreement of a novel Zenit RA chemiluminescent immunoassay (CLIA) from A. Menarini Diagnostics (Florence, Italy) and the gold standard immunoprecipitation assay to screen for the presence of specific anti-U1snRNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo-1(histRNA-Synthetase) and anti-Scl-70(Topo I) antibodies.
Materials and methods
We studied 114 sera, 98 from patients with well-defined autoimmune connective tissue diseases and 16 from blood donor volunteers. All samples were fully characterized using the new chemiluminescent immunoassay and immunoprecipitation. In addition, all the samples were analyzed by indirect immunofluorescence (IIF) and anti-Scl-70(Topo I) antibodies were analyzed by immunoblot (IB) assay. Discrepant samples were analyzed using a commercial dot blot technique (Recomline from Mikrogen). The simple Kappa coefficient was used to measure the level of agreement between the results of Zenit RA CLIA and the gold standard.
The Kappa agreement between Zenit RA CLIA and gold standard immunoprecipitation, as well as IB and IIFassays for the presence of anti-Scl-70(Topo I)(0.948) was excellent. The concordance between Zenit RA CLIA and gold standard immunoprecipitation for the presence of anti-U1snRNP (0.883), anti-Ro/SS-A (0.878), anti-Jo-1(histRNA-Synthetase) (0.791) and anti-Sm (0.786) was good, and excellent when the cut-off was raised to 14 U/ml (arbitrary units/ml). Between Zenit RA CLIA and gold standard immunoprecipitation for the presence of anti-La/SS-B, the Kappa agreement had a value of 0.689, but this improved to 0.775 when the cut-off was raised to14 U/ml. Precision was good based on the evaluation of replicate samples. Inter-assay coefficient variation was lower than 3.4 % (CV in %) in all the kits and <1.2 % (CV in  %) for intra-assay measurements.
Our findings show that Zenit RA CLIA was specific and sensitive to detect anti-U1snRNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo-1(histRNA-Synthetase) and anti-Scl70(Topo I) autoantibodies. This simple, fast and precise method can be a suitable option to analyze these autoantibody specificities.
PMCID: PMC4389043  PMID: 26000155
Specificity; Sensitivity; Zenit RA chemiluminescent immunoassay; Immunoprecipitation; U1snRNP; Sm; Ro/SS-A; La/SS-B; Jo1(histRNA-synthetase); Scl-70(Topo-I); Autoantibodies; ENA
17.  Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry 
Clinics  2013;68(11):1469-1473.
High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages.
A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, β-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α−L-iduronidase activities. Additionally, 13 affected patients were analyzed.
The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls.
Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses.
PMCID: PMC3812554  PMID: 24270961
Dried Blood Spot Analysis; Pompe Disease; Fabry Disease; Gaucher Disease; Krabbe Disease
18.  Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue 
BMC Genetics  2013;14:105.
Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis.
All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort.
Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples.
gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies.
PMCID: PMC3817355  PMID: 24168095
Premature newborns; Single nucleotide polymorphism; Newborn dried blood spots; Umbilical cord; Genomic DNA; Pyrosequencing; Sample storage
19.  Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population 
Screening for carriers of spinal muscular atrophy (SMA) is necessary for effective clinical/prenatal diagnosis and genetic counseling. However, a population-based study of SMA prevalence in mainland China has not yet been conducted. In this study, the copy number of survival motor neuron (SMN) genes was determined in 1712 newborn cord blood samples collected from southern China and from 25 core families, which included 26 SMA patients and 44 parents, to identify SMA carriers. The results presented 13 groups with different SMN1/SMN2 ratios among 1712 newborn individuals, which corresponded to 1535 subjects with two copies of SMN1, 119 with three copies of SMN1, 17 with four copies of SMN1, and 41 with a heterozygous deletion of SMN1 exon 7. Simultaneously, two ‘2+0' genotypes and two point mutations were found among the 44 obligate carriers in the core families, including a novel SMN1 splice-site mutation that was identified in the junction between intron 6 and exon 7 (c. 835–1G>A). These results indicated that the carrier frequency is 1/42 in the general Chinese population and that duplicated SMN1 alleles and de novo deletion mutations are present in a small number of SMA carriers. In addition, we developed and validated a new alternative screening method using a reverse dot blot assay for rapid genotyping of deletional SMA. Our research elucidated the genetic load and SMN gene variants that are present in the Chinese population, and could serve as the basis for a nationwide program of genetic counseling and clinical/prenatal diagnosis to prevent SMA in China.
PMCID: PMC2987421  PMID: 20442745
SMA; SMN copy number; carrier screening; RDB; DHPLC
20.  High Prevalence of Vitamin D Deficiency in Native versus Migrant Mothers and Newborns in the North of Italy: A Call to Act with a Stronger Prevention Program 
PLoS ONE  2015;10(6):e0129586.
Vitamin D status during pregnancy is related to neonatal vitamin D status. Vitamin D deficiency has been associated with an increased risk of rickets in children and osteomalacia in adults. Aim of this study was to investigate 25OHD levels in maternal serum and in neonatal blood spots in native and migrant populations living in Novara (North Italy, 45°N latitude).
Methods and Findings
We carried out a cross sectional study from April 1st 2012 to March 30th 2013, in a tertiary Care Center. Maternal blood samples after delivery and newborns' blood spots were analyzed for 25OHD levels in 533 pairs. Maternal country of origin, skin phototype, vitamin D dietary intake and supplementation during pregnancy were recorded. Multivariate regression analysis, showed a link between neonatal and maternal 25OHD levels (R-square:0.664). Severely deficient 25OHD values (<25 nmol/L) were found in 38% of Italian and in 76.2% of migrant’s newborns (p <0.0001), and in 18% of Italian and 48,4% of migrant mothers (p <0.0001) while 25OHD deficiency (≥25 and <50 nmol/L) was shown in 40.1% of Italian and 21.7% of migrant’s newborns (p <0.0001), and in 43.6% of Italian and 41.3% of migrant mothers (p <0.0001). Italian newborns and mothers had higher 25OHD levels (34.4±19.2 and 44.9±21.2nmol/L) than migrants (17.7±13.7 and 29.7±16.5nmol/L; p<0.0001). A linear decrease of 25OHD levels was found with increasing skin pigmentation (phototype I 42.1 ±18.2 vs phototype VI 17.9±10.1 nmol/l; p<0.0001). Vitamin D supplementation resulted in higher 25OHD values both in mothers and in their newborns (p<0.0001).
Vitamin D insufficiency in pregnancy and in newborns is frequent especially among migrants. A prevention program in Piedmont should urgently be considered and people identified as being at risk should be closely monitored. Vitamin D supplementation should be taken into account when considering a preventative health care policy.
PMCID: PMC4466139  PMID: 26067469
21.  Newborn Screening for Hunter Disease: A Small-Scale Feasibility Study 
JIMD Reports  2013;14:23-27.
Hunter disease (Mucopolysaccharidosis type II, MPS II) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase (IDS). Two main therapies have been reported for MPS II patients: enzyme-replacement therapy (ERT) and hematopoietic stem-cell transplantation (HSCT). Both treatment modalities have been shown to improve some symptoms, but the results with regard to cognitive functioning have been poor. Early initiation of therapy, i.e., before neurological symptoms have manifested, may alter cognitive outcome. The need for early identification makes Hunter disease a candidate for newborn screening (NBS). Our objective was to explore the use of a fluorometric assay that could be applicable for high-throughput analysis of IDS activity in dried blood spots (DBS). The median IDS activity in DBS samples from 1,426 newborns was 377 pmol/punch/17 h (range 78–1111). The IDS activity in one sample was repeatedly under the cutoff value (set at 20% of the median value), which would imply a recall rate of 0.07%. A sample from a clinically diagnosed MPS II individual, included in each 96-well test plate, had IDS activities well below the 20% cutoff value. Coefficients of variation in quality control samples with low, medium, and high IDS activities (190, 304, and 430 pmol/punch/17 h, respectively) were 12% to 16%. This small-scale pilot study shows that newborn screening for Hunter disease using a fluorometric assay in DBS is technically feasible with a fairly low recall rate. NBS may allow for identification of infants with Hunter disease before clinical symptoms become evident enabling early intervention.
PMCID: PMC4213338  PMID: 24272678
22.  Factors associated with knowledge of and satisfaction with newborn screening education: a survey of mothers 
Genetics in Medicine  2012;14(12):963-970.
Effective parental education about newborn blood-spot screening may facilitate prompt follow-up, reduce psychosocial harms, and promote trust in screening programs. However, little is known about the aspects of education delivery and content that are of most importance for fostering understanding and meeting parental expectations. We aimed to identify elements of newborn blood-spot screening education and their associations with mothers' knowledge and satisfaction levels.
We conducted a survey (by mail) of 1,712 mothers who were residing in Ontario, Canada, and whose infants had recently undergone newborn blood-spot screening.
We received 750 completed questionnaires (response rate 47%). Factors associated with respondents' higher knowledge of newborn blood-spot screening were higher level of education (odds ratio = 2.79), English being spoken at home (odds ratio = 1.96), receiving an information sheet at the time of newborn blood-spot screening (odds ratio = 1.57), and receiving information about how to interpret the results (odds ratio = 2.65). Factors associated with being satisfied were: receiving information prenatally (odds ratio = 2.35), from a health-care professional (odds ratio = 4.54), or from an information sheet at the time of newborn blood-spot screening (odds ratio = 1.72); and receiving messages about the purpose of screening (odds ratio = 3.78), the communication process (odds ratio = 2.57), the interpretation of the results (odds ratio = 4.19), and sample-handling methods (odds ratio = 3.13).
Promoting mothers' understanding and meeting their expectations with respect to education about newborn blood-spot screening may require greater engagement with prenatal providers. It also calls for a greater emphasis on communicating with mothers about how blood samples are handled and about the meaning of the test results.
PMCID: PMC3908555  PMID: 22899093
education; genetic screening; newborn screening; public health genomics
23.  Archived unfrozen neonatal blood spots are amenable to quantitative gene expression analysis 
Neonatology  2008;95(3):210-216.
State laws in the US mandate that blood be drawn from all newborn infants to screen for health-threatening conditions. These screening assays consume only a small portion of the blood samples, which are collected on filter paper (“Guthrie”) cards. Many states archive unused blood spots, often in unrefrigerated storage.
While individual RNA transcripts have been identified from archived neonatal blood spots, no study to date has performed quantitative analysis of archived blood spot RNA.
We demonstrate that RNA can be isolated and amplified from newborn blood spots stored unfrozen for as long as nine years, and can be analyzed by microarray and qPCR.
Microarray assays of archived neonatal blood spots consistently detected 3,000-4,000 expressed genes with correlations of .90 between replicates. Blood spot mRNA is amenable to qPCR and we detected biologically relevant expression levels of housekeeping and immune-mediating genes.
These experiments demonstrate the feasibility of using blood spots as a source of RNA which can be analyzed using quantitative microarray and qPCR assays. The application of these methods to the analysis of widely collected biological specimens may be a valuable resource for the study of perinatal determinants of disease development.
PMCID: PMC2693916  PMID: 18799893
Guthrie; Blood Spot; RNA; Microarray; qPCR; Real-Time PCR; Neonatal Screening
24.  Archived Unfrozen Neonatal Blood Spots Are Amenable to Quantitative Gene Expression Analysis 
Neonatology  2008;95(3):210-216.
State laws in the USA mandate that blood be drawn from all newborn infants to screen for health-threatening conditions. These screening assays consume only a small portion of the blood samples, which are collected on filter paper (‘Guthrie’) cards. Many states archive unused blood spots, often in unrefrigerated storage. Objectives: While individual RNA transcripts have been identified from archived neonatal blood spots, no study to date has performed quantitative analysis of archived blood spot RNA.
We demonstrate that RNA can be isolated and amplified from newborn blood spots stored unfrozen for as long as 9 years, and can be analyzed by microarray and qPCR.
Microarray assays of archived neonatal blood spots consistently detected 3,000–4,000 expressed genes with correlations of 0.90 between replicates. Blood spot mRNA is amenable to qPCR and we detected biologically relevant expression levels of housekeeping and immune-mediating genes.
These experiments demonstrate the feasibility of using blood spots as a source of RNA which can be analyzed using quantitative microarray and qPCR assays. The application of these methods to the analysis of widely collected biological specimens may be a valuable resource for the study of perinatal determinants of disease development.
PMCID: PMC2693916  PMID: 18799893
Guthrie; Blood spot; RNA; Microarray; qPCR; Real-time PCR; Neonatal screening
25.  The first case of mitochondrial acetoacetyl-CoA thiolase deficiency identified by expanded newborn metabolic screening in Italy: the importance of an integrated diagnostic approach 
Journal of Inherited Metabolic Disease  2010;33(Suppl 3):91-94.
A pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) began in the Campania region, southern Italy, in 2007. By October 2009, >8,800 dried blood samples on filter paper from 11 hospitals had been screened. Within this screening programme, we identified a case of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency [β-ketothiolase (β-KT) deficiency] by analysing the acylcarnitine profile from a dried blood spot with LC-MS/MS. Gas chromatography coupled with mass spectrometry analysis of urinary organic acids and LC-MS/MS analysis of urinary acylcarnitines were in line with this disorder. In fact, concentrations were well beyond the cut-off values of tiglyl carnitine, 3-hydroxybutyrylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine, 2-methyl-3-hydroxybutyric acid and tiglyl glycine. The absence of 2-methylacetoacetic acid in urine may be attributed to: (i) the instability of this β-ketoacid because it undergoes spontaneous decarboxylation to 2-butanone, which is highly volatile and thus difficult to detect, and (ii) the good health of the patient in the first days of life. β-KT deficiency was subsequently diagnosed in the patient's older sister, who showed increased levels of the same metabolites but also small amounts of 2-methylacetoacetic acid, which is considered a key marker for β-KT diagnosis. Genomic analysis revealed mutation c.1189C >G in exon 12 of the ACAT1 gene, which results in a severe defect because of the p.H397D amino acid change in both alleles of both patients.
PMCID: PMC3757262

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