MSACL 2025 Abstract

Improving Urinary Newborn Screening Second-Tier Test via Mass Spectrometry for Enhanced Quality Assessment

Floresse Kpaossou, Tristan Martineau, Marie-Claude Déry, Pamela Lavoie, Christiane Auray-Blais
Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, QC, Canada J1H 5N4, Université de Sherbrooke

Floresse Kpaossou, Bachelor's degree in Biochemistry (Presenter) Université de Sherbrooke

Presenter Bio: Floresse Kpaossou holds a bachelor's degree in biochemistry and works as a research assistant at the Université de Sherbrooke. Her current work focuses on quantitative analysis of Lyso-Gb3 in plasma and urine, and the development of a second-tier test method for newborn screening using mass spectrometry. With a growing interest in clinical applications of mass spec, Floresse is eager to explore method development and biomarker discovery.

Relevant Financial Disclosures (within past 24 months, reported on May 26, 2025) No relevant financial relationship(s) to disclose.

INTRODUCTION
Newborn screening (NBS) is part of an important public health program that enables the early detection of inborn errors of metabolism (IEMs), helping to prevent severe clinical complications and improve patients’ outcomes. In Quebec, urine-based NBS has traditionally relied on thin-layer chromatography (TLC) using dried urine spots. However, TLC has limitations in sensitivity and specificity, prompting efforts to transition to tandem mass spectrometry (UPLC-MS/MS) for both primary screening and second-tier testing. A key component of this transition is the establishment of normal reference values for specific biomarkers to ensure accurate differentiation between healthy and affected newborns. Additionally, maintaining the integrity of the test results requires robust quality control measures to detect potential urine filter paper sample contamination. Expanding the existing second-tier test method to include additional key compounds will further enhance its reliability, accuracy, and facilitate the overall interpretation of results.

OBJECTIVES
The objectives of this research project were: 1) to improve the existing second-tier testing method by adding new compounds to assess the integrity of filter paper and urine samples, establish good quality controls, and enhance the test reliability; 2) establish normal UPLC-MS/MS reference values for the key NBS biomarkers while ensuring accurate differentiation between healthy and affected newborns.

METHODS
At 21 days of age, parents collect urine specimens using a standardized collection kit, which are then dried onto filter paper and mailed to the laboratory. Upon arrival, the samples undergo an initial quality check under ultraviolet (UV) light to assess the quantity of urine and identify potential contaminants. The samples are presently analyzed qualitatively using TLC for amino acids and organic acids. A research evaluative upgrade of the existing TLC method to mass spectrometry has led to the establishment of second-tier test for the eluted urine filter paper samples. The samples are diluted then analysed using an Acquity I-Class UPLC system coupled to a Xevo TQ-S Micro tandem mass spectrometer (Waters Corp.). The UPLC-MS/MS analysis is performed using a BEH C18 column AX 1.7 µm, 2.1x100 mm column (Waters Corp.), utilizing both positive and negative ionization modes.

RESULTS
Eight new compounds (adipic, fumaric, hippuric, lactic, oxalic, pyroglutamic, sebacic, suberic acids) were successfully added to the 16 biomarkers already analysed (glutaric acid, N-isovalerylglycine, 2-methylcitric acid, methylmalonic acid, propionylglycine, 3-hydroxyisovaleric acid, 3-propionic acid, 3-methylcrotonylglycine, malonic acid, orotic acid, 3-hydroxyglutaric acid, homogentisic acid, guanidinoacetate, creatine, creatinine, uracil) in the existing UPLC-MS/MS second-tier test method. As of now, a total of 550 control samples have been analyzed to establish normal reference values for the 16 initial biomarkers in the method, while 138 control samples were analyzed for the 8 additional compounds. The addition of these compounds improved the detection of urine contamination due to diet or certain medications, further enhancing the reliability of test results. The normal reference values established allowed for a clear distinction between healthy and affected newborns.

CONCLUSION
This study demonstrates the successful integration of UPLC-MS/MS into newborn screening for second-tier testing providing a significant improvement over traditional TLC-based method. Establishing reference values for key biomarkers enhances diagnostic accuracy and ensures more reliable differentiation between healthy and affected newborns. Furthermore, the addition of new biomarkers for contamination detection strengthens quality controls, reducing potential issues related to sample integrity, and facilitates the overall interpretation of results. These advancements pave the way for a more efficient and accurate newborn screening program, ultimately leading to earlier detection, fewer false positives, and better patient outcomes.