Integrated Multianalyte Second-Tier Testing for Newborn Screening for MSUD, IVA, and GAMT Deficiencies

Abstract

Advances in mass spectrometry have allowed for expansion of newborn screening test panels over the last decade but with increased numbers of disorders have come increased concerns with false-positive rates. The introduction of second-tier testing has improved the specificity of screening for a number of disorders without any corresponding sacrifice in sensitivity. Such testing does, however, put pressure on scarce laboratory resources including instrument and personnel time and even the bloodspot sample itself. The British Columbia Newborn Screening Program has developed an integrated second-tier screening approach to improve test performance without the requirement to resample and reprocess the original bloodspot specimen. By utilizing the residual extract from the first-tier assay and introducing a chromatography step as the second tier, we have been able to reduce false-positive rates due to interfering isobaric compounds for 3 different disorders (maple syrup urine disease, isovaleric aciduria, and guanidinoacetate methyltransferase) in a single multianalyte assay.

Quantitative Determination of Branched-Chain Amino Acids in Dried Blood Spot Samples by LC-MSMS and its Application in Diagnosis and Follow-Up of Chilean Patients with Maple Syrup Urine Disease

“…Retrospective analysis by LC-MSMS of samples of MSUD patients revealed that all of them presented quantifiable levels of Allo with values above the upper COV reported in literature for Allo (2-5 µmol/L). [13,14,23] Allo seems to be stable over time since it was detected even in samples that were stored for more than five-years in a non-controlled environment. Thus, it appears to be a highly sensitive biomarker for samples taken as early as 24h of life (7.1 µmol/L), as was the case for the MSUD patient diagnosed by family history.…”

“…The introduction of MSUD second-tier testing in the NBS program in British Columbia, Canada showed that all falsepositive samples (n=7) presented normal values of Allo-Ile (<2 µmol/L) whereas all true positives (n=3) had markedly higher levels of Allo-Ile (>22 µmol/L). [14] Although Allo-Ile is a wellrecognized biomarker for MSUD, in an intermittent MSUD phenotype, Allo elevation may not be present in NBS samples, but its formation and detection can be observed under catabolic state. [24] Also, it has been reported that elevated levels of Allo and Leu may be found in PKU patient's (6 of 11 patients studied) due to liver damage which could lead to false positive result.…”

“…The method consists in separating each isobaric form of XLeu and quantifying them independently using liquid chromatography-tandem mass spectrometry (LC-MSMS) in the original DBS sample used for first test. [7,[13][14][15][16] As early as in the first 12 to 24 hours of life, MSUD newborns exhibit elevated plasma concentration of BCAAs Leu, Ile and Val together with Allo, undetected in normal individuals, making this the pathognomonic marker of disease [17] and required for confirmatory diagnosis. [18] Several protocols for BCAAs and Allo quantification in DBS have been published and have demonstrated subtle differences that make implementation feasible for specialized metabolic laboratories.…”

“…[18] Several protocols for BCAAs and Allo quantification in DBS have been published and have demonstrated subtle differences that make implementation feasible for specialized metabolic laboratories. [13][14][15][16] Outside of its initial application as a second-tier test for MSUD, the LC-MSMS method for BCAAs quantification in DBS samples may be useful for rapid confirmatory diagnosis when MSUD patients are diagnosed by clinical findings or by family history due to sibling diagnosis, as generally happens in nations like Chile and most of South American countries where the local NBS program does not include MSUD. Detection and quantification of Allo in DBS samples of potential MSUD cases, in conjunction with determination of Leu/Phe ratio would be a useful approach for shortening the time during the confirmatory diagnosis process.…”

Retrospective Review of Positive Newborn Screening Results for Isovaleric Acidemia and Development of a Strategy to Improve the Efficacy of Newborn Screening in the UK

“…This would successfully address the problem of false positives due to pivalate and prevent the unnecessary referral of these babies. Second-tier testing protocols are becoming increasingly common in screening programs around the world and enabled additional disorders, e.g., disorders of propionate metabolism, classical homocystinuria and remethylation disorders, maple syrup urine disease, guanidinoacetate methyl transferase deficiency, to be included in existing programs whilst minimizing FP rates and improving the efficacy of screening [ 15 , 16 , 17 , 18 , 19 ]. There are, however, practical issues to consider in this context, as screen-positive results for IVA are relatively rare.…”