A rapid UPLC–MS/MS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening

2012 ◽  
Vol 404 (3) ◽  
pp. 741-751 ◽  
Author(s):  
Antonina Gucciardi ◽  
Paola Pirillo ◽  
Iole Maria Di Gangi ◽  
Mauro Naturale ◽  
Giuseppe Giordano
Keyword(s):  
Newborn Screening ◽  
Dried Blood Spots ◽  
Blood Spots ◽  
Expanded Newborn Screening ◽  
Simultaneous Separation ◽  
Second Tier

scholarly journals Second-Tier Test for Quantification of Alloisoleucine and Branched-Chain Amino Acids in Dried Blood Spots to Improve Newborn Screening for Maple Syrup Urine Disease (MSUD)

2008 ◽  
Vol 54 (3) ◽  
pp. 542-549 ◽  
Author(s):  
Devin Oglesbee ◽  
Karen A Sanders ◽  
Jean M Lacey ◽  
Mark J Magera ◽  
Bruno Casetta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Newborn Screening ◽  
Dried Blood Spots ◽  
Branched Chain Amino Acids ◽  
Maple Syrup ◽  
Blood Spots ◽  
Urine Disease ◽  
Second Tier ◽  
Branched Chain ◽  
Positive Results

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

scholarly journals Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses

2020 ◽  
Vol 6 (3) ◽  
pp. 51 ◽  
Author(s):  
Trine Tangeraas ◽  
Ingjerd Sæves ◽  
Claus Klingenberg ◽  
Jens Jørgensen ◽  
Erle Kristensen ◽  
...  
Keyword(s):  
Newborn Screening ◽  
Screening Program ◽  
Good Health ◽  
Dried Blood Spots ◽  
Case Definitions ◽  
Inborn Errors ◽  
Disease Spectrum ◽  
Expanded Newborn Screening ◽  
Second Tier ◽  
Biochemical Testing

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies.

Next-generation sequencing as a second-tier diagnostic test for newborn screening

2018 ◽  
Vol 31 (8) ◽  
pp. 927-931 ◽  
Author(s):  
Xiaomei Luo ◽  
Ruifang Wang ◽  
Yanjie Fan ◽  
Xuefan Gu ◽  
Yongguo Yu
Keyword(s):  
Next Generation Sequencing ◽  
Newborn Screening ◽  
Diagnostic Test ◽  
Genomic Dna ◽  
Sanger Sequencing ◽  
Dried Blood Spots ◽  
Next Generation ◽  
Blood Spots ◽  
Second Tier ◽  
Generation Sequencing

Abstract Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.

Newborn screening for galactosemia by a second-tier multiplex enzyme assay using UPLC-MS/MS in dried blood spots

2011 ◽  
Vol 34 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Dae-Hyun Ko ◽  
Sun-Hee Jun ◽  
Kyoung Un Park ◽  
Sang Hoon Song ◽  
Jin Q Kim ◽  
...  
Keyword(s):  
Newborn Screening ◽  
Enzyme Assay ◽  
Dried Blood Spots ◽  
Blood Spots ◽  
Second Tier

scholarly journals Quantification of Differential Metabolites in Dried Blood Spots Using Second-Tier Testing for SCADD/IBDD Disorders Based on Large-Scale Newborn Screening in a Chinese Population

2021 ◽  
Vol 9 ◽  
Author(s):  
Wei Zhou ◽  
Heng Cai ◽  
Huizhong Li ◽  
Zhe Ji ◽  
Maosheng Gu
Keyword(s):  
Newborn Screening ◽  
Large Scale ◽  
Dehydrogenase Deficiency ◽  
Clinical Performance ◽  
False Positive Rate ◽  
Dried Blood Spots ◽  
Care Hospital ◽  
Blood Spots ◽  
Metabolic Defects ◽  
Second Tier

Background: Although newborn screening (NBS) for metabolic defects using the marker butyl carnitine (C4) combined with the C4-to-acetylcarnitine ratio is adequate, the incorporation of novel parameters may improve differential testing for these disorders without compromising sensitivity.Methods: Analytical and clinical performance was evaluated by MS/MS using 237 initially positive neonatal samples between March 2019 and March 2020 at the Newborn Screening Center of Xuzhou Maternity and Child Health Care Hospital. Additionally, second-tier testing by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with the quantification of ethylmalonate (EMA) or isobutyryl-glycine (IBG) in dried blood spots (DBSs) was performed to reduce the false-positive rate.Results: We reviewed initial MS/MS data for DBSs from 469,730 neonates, and a second-tier test was performed using 237 samples that exceeded the C4 concentration cutoff value. Eleven variants of the ACADS gene were identified, with c.1031A>G (p.E344G) being the most common. Fifteen ACAD8 mutations were identified in seven patients, and Swiss modeling and amino acid conservation analyses were conducted for the novel variants. Based on a retrospective analysis of EMA and IBG, the application of second-tier tests before the release of neonatal screening results reduced referrals by over 91.89% and improved the positive predictive value (PPV) for short-chain acyl-CoA dehydrogenase deficiency/isobutyryl-CoA dehydrogenase deficiency (SCADD/IBDD) screening.Conclusion: A screening algorithm including EMA/IBG improves target differential testing for NBS and may eliminate unnecessary referrals while maintaining 100% sensitivity. Second-tier screening using UPLC-MS/MS as a rapid and convenient supplemental DNA sequencing method may be beneficial for differential detection.

scholarly journals QUALITY OF DRIED BLOOD SPOTS IS AN INTEGRAL COMPONENT OF PROMPT DETECTION OF INBORN ERRORS OF METABOLISM

2020 ◽  
Vol 10 (4(38)) ◽  
pp. 77-86
Author(s):  
Tetiana Znamenska ◽  
O. Vorobiova ◽  
I. Kuzneczov ◽  
I. Lastivka ◽  
A. Kremezna ◽  
...  
Keyword(s):  
Newborn Screening ◽  
Inborn Errors Of Metabolism ◽  
Dried Blood Spots ◽  
Poor Quality ◽  
Blood Sampling ◽  
Inborn Errors ◽  
Blood Spots ◽  
Expanded Newborn Screening ◽  
Practical Recommendations

Introduction. Inborn Errors of Metabolism (IEM) are constituted a group of genetic diseases that are associated with defects in the synthesis or catabolism of complex molecules, impaired intermediary metabolism and energy production/utilization processes. The clinical manifestation of IEM is nonspecific, that looks similar to septicemia, and most often occurs in the neonatal period with life-threatening acute metabolic crises. Expanded Newborn Screening (ENS) – a biochemical study of the blood of all newborns without exception with the purpose to identify molecular markers of these diseases proved to be the most effective instrument of early IEM diagnostics. The quality of the biological samples (dried blood spots, DBS) in great extent determines the timing, accuracy, and reliability of the results of biochemical measurements. Obtaining of equivocal results in the case of analysis of poor quality DBS requires repeated laboratory tests, that delays the diagnostic process and postpones the start of specific treatment, which usually results in irreversible damage of the brain and internal organs of the child. The aim of this work is to (i) review the first results of the implementation of Expanded Newborn Screening in Ukraine (pilot part of the Baby Screen Project), and to analyze literature data regarding the negative impact of poor quality DBS on laboratory determination of IEM marker substances contents in the specimens, (ii) to characterize the typical errors in blood sampling and drying of  blood spots, and (iii) to provide practical recommendations for the proper performance of these procedures. Materials and methods. Own data of retrospective analysis of the questionable ENS results was superimposed with dried blood specimens, that were investigated in the Pharmbiotest ENS Lab to outline most common inaccuracies. Based on the comparison of these data with the relevant publications it was formulated the practical recommendations for improving quality of DBS preparation to ensure the accuracy and reliability of laboratory measurements and speed up IEM diagnostics. Results. The quality of biomaterial selection is an important part of obtaining reliable results during expanded newborn screening. Capillary blood is collected in the maternity hospital from 48-72 hours (full-term) and for 7-11 days (in preterm babies) after the birth from the heel of babies. In this case, a few drops of blood are applied to a special test card made of filter paper, which is dried and sent to the laboratory. Blood tests are performed using a highly sensitive and accurate method of chemical analysis - tandem mass spectrometry in the laboratory "Pharmbiotest", located in Ukraine. Taking into analysis the low-quality samples lead to questionable results, which requires repeated DBS sampling and re-examining. This proved to be the most common cause of delaying IEM detection, diagnosis establishment, and initiation of treatment, which can be fatal for a child with severe IEM forms. Conclusions. Informing healthcare professionals and parents about the current results of laboratory monitoring of dried blood spots quality, typical errors in blood sampling and following on-site procedures and negative consequences of its improper performance, as well as providing clear practical recommendations of how these procedures should be done is a proven way of improving and speeding up IEM diagnostics.

scholarly journals Cystic Fibrosis Newborn Screening in Austria Using PAP and the Numeric Product of PAP and IRT Concentrations as Second-Tier Parameters

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 299
Author(s):  
Maximilian Zeyda ◽  
Andrea Schanzer ◽  
Pavel Basek ◽  
Vera Bauer ◽  
Ernst Eber ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Newborn Screening ◽  
Dried Blood Spots ◽  
False Negatives ◽  
Blood Spots ◽  
Second Tier ◽  
Pancreatitis Associated Protein ◽  
Immunoreactive Trypsinogen ◽  
Sweat Tests ◽  
Observation Period

In Austria, newborns have been screened for cystic fibrosis (CF) by analyzing immunoreactive trypsinogen (IRT) from dried blood spots (DBS)s for nearly 20 years. Recently, pancreatitis-associated protein (PAP) analysis was introduced as a second-tier test with the aim of reducing recalls for second DBS cards while keeping sensitivity high. For 28 months, when IRT was elevated (65–130 ng/mL), PAP was measured from the first DBS (n = 198,927) with a two-step cut-off applied. For the last 12 months of the observation period (n = 85,421), an additional IRT×PAP cut-off was introduced. If PAP or IRT×PAP were above cut-off, a second card was analyzed for IRT and in case of elevated values identified as screen-positive. Above 130 ng/mL IRT in the first DBS, newborns were classified as screen-positive. IRT analysis of first DBS resulted in 1961 (1%) tests for PAP. In the first 16 months, 26 of 93 screen-positive were confirmed to have CF. Two false-negatives have been reported (sensitivity = 92.8%). Importantly, less than 30% of families compared to the previous IRT-IRT screening scheme had to be contacted causing distress. Adding IRT×PAP caused a marginally increased number of second cards and sweat tests to be requested during this period (15 and 3, respectively) compared to the initial IRT-PAP scheme. One case of confirmed CF was found due to IRT×PAP, demonstrating an increase in sensitivity. Thus, the relatively simple and economical algorithm presented here performs effectively and may be a useful model for inclusion of CF into NBS panels or modification of existing schemes.

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