Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

In 2015, Pompe disease became the first lysosomal storage disorder to be recommended for universal newborn screening by the Secretary of the U.S. Department of Health and Human Services. Newborn screening for Pompe has been implemented in 20 states and several countries across the world. The rates of later-onset disease phenotypes for Pompe and pseudodeficiency alleles are higher than initially anticipated, and these factors must be considered during Pompe disease newborn screening. This report presents an overview of six years of data from the Missouri State Public Health Laboratory for Pompe disease newborn screening and follow-up.

Krabbe disease (KD) is part of newborn screening (NBS) in 11 states with at least one additional state preparing to screen. In July 2021, KD was re-nominated for addition to the federal Recommended Uniform Screening Panel (RUSP) in the USA with a two-tiered strategy based on psychosine (PSY) as the determinant if an NBS result is positive or negative after a first-tier test revealed decreased galactocerebrosidase activity. Nine states currently screening for KD include PSY analysis in their screening strategy. However, the nomination was rejected in February 2023 because of perceived concerns about a high false positive rate, potential harm to newborns with an uncertain prognosis, and inadequate data on presymptomatic treatment benefit or harm. To address the concern about false positive NBS results, a survey was conducted of the eight NBS programs that use PSY and have been screening for KD for at least 1 year. Seven of eight states responded. We found that: (1) the use of PSY is variable; (2) when modeling the data based on the recommended screening strategy for KD, and applying different cutoffs for PSY, each state could virtually eliminate false positive results without major impact on sensitivity; (3) the reason for the diverse strategies appears to be primarily the difficulty of state programs to adjust screening algorithms due to the concern of possibly missing even an adult-onset case following a change that focuses on infantile and early infantile KD. Contracts with outside vendors and the effort/cost of making changes to a program’s information systems can be additional obstacles. We recommend that programs review their historical NBS outcomes for KD with their advisory committees and make transparent decisions on whether to accept false positive results for such a devastating condition or to adjust their procedures to ensure an efficient, effective, and manageable NBS program for KD.

Mucopolysaccharidosis Type II (MPS II), also known as Hunter syndrome, is a lysosomal storage disorder (LSD) caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). MPS II satisfies all criteria defined by the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) for inclusion in the Recommended Uniform Screening Panel (RUSP) for newborn screening, apart from the fact that only minimal prospective population screening data are available. This report details the analytical validation, clinical validation, and implementation of a fluorometric assay for measurement of IDS activity in newborn dried blood spot (DBS) specimens at the Missouri State Public Health Laboratory (MSPHL). The assay is performed in a microwell plate format requiring approximately 15 min of hands-on time per plate and an incubation time of two hours. The analytical validation of this assay included linearity, analytical sensitivity, precision, and carry-over testing. Clinical validation was completed using more than 5000 deidentified presumptive normal newborn DBS specimens as well as seven specimens from patients known to be affected with MPS II. Following validation, MSPHL began prospective screening using the IDS assay on 1 November 2018. In the first 18 months of screening (to 30 June 2020), 146,954 specimens were prospectively screened using the method. Two newborns were identified with severe Hunter syndrome and the assay had a presumptive positive rate of 0.022%.

In newborn screening, false-negative results can be disastrous, leading to disability and death, while false-positive results contribute to parental anxiety and unnecessary follow-ups. Cutoffs are set conservatively to prevent missed cases for Pompe and MPS I, resulting in increased falsepositive results and lower positive predictive values. Harmonization has been proposed as a way to minimize false-negative and false-positive results and correct for method differences, so we harmonized enzyme activities for Pompe and MPS I across laboratories and testing methods (Tandem Mass Spectrometry (MS/MS) or Digital Microfluidics (DMF)). Participating states analyzed proofof- concept calibrators, blanks, and contrived specimens and reported enzyme activities, cutoffs, and other testing parameters to Tennessee. Regression and multiples of the median were used to harmonize the data. We observed varied cutoffs and results. Six of seven MS/MS labs reported enzyme activities for one specimen for MPS I marginally above their respective cutoffs with results classified as negative, whereas all DMF labs reported this specimen’s enzyme activity below their respective cutoffs with results classified as positive. Reasonable agreement in enzyme activities and cutoffs was achieved with harmonization; however, harmonization does not change how a value would be reported as this is dependent on the placement of cutoffs.

Newborn screening laboratories are increasingly adding lysosomal storage disorders (LSDs), such as Mucopolysaccharidosis I (MPS I) and Pompe disease, to their screening panels. Without newborn screening, LSDs are frequently diagnosed only after the onset of symptoms; late detection can lead to profound and irreversible organ damage and mortality. While screening of these disorders has accelerated over the past five years, there is little published information regarding the potential correlation of demographic variables (age at sample collection, birthweight, gestational age, gender, etc.) with lysosomal enzyme activity. The Missouri State Public Health Laboratory prospectively screened more than 475,000 newborns for MPS I, Pompe disease, Gaucher disease, and Fabry disease between 15 January 2013 and 15 May 2018. This report investigates trends between several demographic variables and activities of four lysosomal enzymes: α-L-iduronidase (IDUA), acid α-glucosidase (GAA), acid β-glucocerebrosidase (GBA), and acid α-galactosidase (GLA). This information provides a valuable resource to newborn screening laboratories for the implementation of screening for lysosomal storage disorders and the establishment of screening cutoffs.