multiplexed separations for rapid confirmatory testing of screen-positive inborn errors of...
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and the combined leucine/isoleucine peak. Residual extracts from allMSUD flagged specimens are then reinjected with a 5 minutechromatographic separation to quantitate the individual branched-chain isobars (Leu, Ile, and Allo-ile) as the second-tier test.
Results: 134,537 newborns have been screened with this methodwith 3 confirmed MSUD cases from 9 positive screen results(PPV = 33%). Two of these infants had a challenging neonatal courserequiring hemodialysis upon admission but both are withoutneurological sequelae at 1 year of follow-up. The third affectedinfant was born with an unrelated cardiac anomaly and wasundergoing surgical repair when the abnormal screening result wasreported. He is also showing normal development at 3 years of age.Dietary management is now being facilitated by frequent bloodspotBCAA monitoring using this method.
Conclusions: This integrated MSUD second-tier screening assayallows for rapid confirmation of abnormal initial screening results,a high positive predictive value, and an efficient tool for therapeuticmonitoring.
doi:10.1016/j.clinbiochem.2014.07.075
Mass spectrometric determination of purine metabolites inDBS: A novel second tier approach to detectingadenosine deaminase deficiency in the screeningfor severe combined immunodeficiencyNathan A. McIntosha, Svetlana Ogrel a, Lawrence Fishera, Michael T.Geraghtya, b, Pranesh Chakrabortya, b, Osama Y. Al-Dirbashia, baNewborn Screening Ontario, Children's Hospital of Eastern Ontario,Ottawa, Ontario, CanadabDepartment of Pediatrics, Faculty of Medicine, University of Ottawa,Ottawa, Ontario, Canada
One-fifth of all cases of severe combined immunodeficiency(SCID) are the result of adenosine deaminase (ADA) deficiencyresulting in the accumulation of toxic metabolites, includingdeoxyadenosine (dAdo), and deoxyadenosine triphosphate. Thesetoxic metabolites cause neurological, hepatic and renal abnormali-ties as well as impairment of the immune function that result in lifethreatening SCID.
At Newborn Screening Ontario, initial screening for SCID involvesthe detection of T –cell receptor excision circles (TREC) in DBS.Samples flagged with abnormally reduced TREC counts are con-firmed in duplicate by analyzing TREC together with a referencegene. Further, these samples are analyzed by a highly specific secondtier MS/MS assay for purine metabolites. These samples are preparedin 96 well plate batches that take approximately 2 h. MS/MSinjection to injection time is 2 min.
Validation of this MS/MS assay was carried out on control DBSsamples (n = 588) with mean adenosine (Ado) and dAdo concen-trations of 2.1 and 0.27 μmol/L, respectively. Samples from individ-uals with ADA deficiency (n = 4) have been tested using this assay,producing concentrations ranging from 21.9 to 33.4 μmol/L of Adoand 40.5–55.2 μmol/L of dAdo.
The serious consequences of ADA deficiency along with thebenefits of timely diagnosis and treatment triggered the develop-ment and implementation of this method. This method unequivocallydetects elevated purine metabolites and facilitates the subsequentdiagnosis and treatment of newbornswith ADAdeficiency. Thismethodwas also able to determine other purine metabolites so that it canbe used for SCID cases caused by purine nucleotide phosphorylasedeficiency.
doi:10.1016/j.clinbiochem.2014.07.076
Multiplexed separations for rapid confirmatory testing ofscreen-positive inborn errors of metabolismAlicia DiBattistaa, Philip Britz-McKibbina,Osama Y. Al-Dirbashib, c, Pranesh Chakrabortyb, c
aMcMaster University, Hamilton, Ontario, CanadabNewborn Screening Ontario, Ottawa, Ontario, CanadacUniversity of Ottawa, Ottawa, Ontario, Canada
Objectives: Direct injection-tandem mass spectrometry forprimary screening of in-born errors of metabolism (IEMs) suffersfrom isobaric/isomeric interferences that require more specificmethods for confirmatory testing of screen-positive results. How-ever, second-tier testing is low-throughput and requires variouschromatographic methods to ensure adequate specificity whenanalyzing different classes of metabolites. Herein, we introducea simple yet high-throughput method for confirmatory testingof IEMs from dried blood spot extracts (DBS) when using multi-segment injection-capillary electrophoresis–mass spectrometry(MSI-CE–MS).
Methods: Methanol extracts of dried blood spots correspondingto five IEMs were analyzed using MSI-CE–MS in conjunction withasymmetric pattern recognition based on an injection-specific patternof sample dilution. Three DBS samples were analyzed in duplicatein a single run alongside a pooled/normal control for unambiguousidentification and quantification of disease-specific markers of IEMswith a sample throughput of about 4 min/sample.
Results: Significantly elevated levels of primary and secondarymetabolite markers were detected and quantified for all IEMsrelative to a control sample. All primary markers and their ratioshad whole blood concentrations above reported diagnostic cut-offvalues for a primary screen. Furthermore, the detection of otherclasses of metabolites differentially expressed in DBS extract samplesmay have diagnostic value for screening IEMs by MS, such as cysticfibrosis.
Conclusions: The use of MSI-CE–MS as a high-throughputconfirmatory testing platform has been validated to identify andquantify elevated levels of primary and secondary markers of IEMs,thus reducing the time and cost associated with chromatographic-based confirmatory testing methods.
doi:10.1016/j.clinbiochem.2014.07.077
Liver cell transplantation as a “bridge” therapy for urea cycledisorders: The Calgary experienceAneal Khana, Steven R. Martinb, Mary Brindlec, Simon Parsonsd,Seemab Haidere, Nicole Prokopishynf, Jason Yapg
aMedical Genetics and Pediatrics, University of Calgary, Alberta Children'sHospital, Calgary, Alberta, CanadabDepartment of Pediatrics, Section of Gastroenterology and Nutrition,Alberta Children's Hospital, Calgary, Alberta, CanadacSurgery, University of Calgary, Alberta Children's Hospital, Calgary,Alberta, CanadadDepartment of Pediatrics, Section of Critical Care, CanadaeCellular Therapy Laboratory, Calgary Lab Services, Calgary,Alberta, CanadafDepartment of Diagnostic Imaging, University of Calgary,Alberta Children's Hospital, Calgary, Alberta, CanadagGastroenterology and Pediatrics, University of Alberta (Hospitals),Edmonton, Alberta, Canada
Objectives: To evaluate liver cell transplantation (LCT) as a“bridge” therapy for urea cycle disorders.
Methods: Open label clinical trial, SELICA III (www.clinicaltrials.gov;NCT01195753) involving 6 days of infusion of blood group-matched
Abstracts150