, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.
Purpose: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. Methods: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. Results: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. Conclusions: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%
A tandem time-of-flight (TOF) mass spectrometer has been designed to obtain complete MS/MS spectra from compounds eluting from a gas chromatograph. This application requires high spectral generation rate, unit mass resolution for both precursor selection and product spectra, and efficient ion utilization. These objectives are achieved by reflectron TOF mass separation in both stages and laser photoinduced dissociation as the ion fragmentation method. Careful timing of the laser pulse relative to ion extraction allows ions of a single m/z value up to m/z 1000 to be photodissociated while ions with adjacent m/z values are essentially unaffected. The convergent foci of the ion packet and laser pulse results in ion fragmentation efficiencies as high as 79%. An ion gate prevents the nonselected precursor ions from convoluting the product spectra. Product spectra can be generated at the maximum laser repetition rate (currently 200 Hz). To achieve unit mass resolution for all product m/z values simultaneously, a novel reflectron was designed for the second TOF stage.
Background 3-hydroxypalmitoleoyl-carnitine (C16:1-OH) was recently reported to be elevated in acylcarnitine profile of propionic acidemia (PA) or methylmalonic acidemia (MMA) patients during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). Methods The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method. A specific retention time was reported for each hydroxylated long chain acylcarnitine. The same method was applied to some neonatal dried blood spots (DBS) from PA and MMA patients presenting abnormal C16:1-OH concentrations. Results The final retention time of the peak corresponding to C16:1-OH in LCHADD patients differed from those in MMA and PA patients. Heptadecanoylcarnitine (C17) has been identified as the novel biomarker specific for PA and MMA patients through high resolution mass spectrometry (Orbitrap) experiments. We found that 21 out of 23 neonates (22 MMA, and 1PA) diagnosed through the Tuscany region NBS program had significantly higher levels of C17 compared to levels detected in controls. Twenty-three maternal deficiencies (21 vitamin B12 deficiency, 1 homocystinuria and 1 gastrin deficiency) and 82 false positive for propionylcarnitine (C3) results were also analyzed. Conclusions This paper reports on the characterization of a novel biomarker able to detect propionate disorders during expanded newborn screening (NBS). The use of this new biomarker may improve the analytical performances of NBS programs especially in laboratories where second tier tests are not performed.
Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD) type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH), α-ketoglutarate dehydrogenase (αKGDH), and pyruvate dehydrogenase (PDH). DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS) metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the “citrullinemia/elevated citrulline” ACMG Act Sheet and Algorithm.
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