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Inclusion of pyridoxine dependent epilepsy in expanded newborn screening programs by tandem mass spectrometry: set up of first and second tier tests

  • Roberta Damiano , Maria Della Bona , Elena Procopio , Renzo Guerrini , Alessandra Bettiol and Giancarlo la Marca ORCID logo EMAIL logo
Published/Copyright: February 4, 2025
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 63 Issue 7

Abstract

Objectives

Pyridoxine-dependent epilepsy (PDE) is a rare genetic disorder characterized by intractable neonatal seizures responsive to pyridoxine. Diagnosis relies on quantification of α-aminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid in urine or plasma in patients with overt symptoms. We developed and validated simple and rapid first- and second-tier methods for two recently published biomarkers of PDE (2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP)) in extended newborn screening (NBS) programs from neonatal dried blood spots (DBS).

Methods

For the first-line test, DBS specimens were collected from 5,405 newborns who underwent routine NBS and analysed by FIA-MS/MS. For the second-tier test, samples were analysed by LC-MS/MS. The neonatal DBS from two patients with genetically confirmed PDE were also analysed.

Results

The reference values for NBS resulted <0.34 μmol/L for 2-OPP and <4.51 μmol/L for 6-oxoPIP. In the second-tier test, limits of detection were 0.07 μmol/L and 0.14 μmol/L, whereas limits of quantification were 0.25 μmol/L and 0.48 μmol/L, respectively, for 2-OPP and for 6-oxoPIP. The tests provided good linearity, reproducibility, accuracy and precision, with acceptable matrix effect and carry-over, according to international validation criteria. The biomarkers in DBS were stable at room temperature, +4 °C and −20 °C for one month. When assessing these biomarkers in two patients with genetically confirmed PDE, the higher sensitivity of 2-OPP as compared to 6-oxoPIP in discriminating PDE emerged.

Conclusions

The first-line and second-tier tests developed in this study highlight the potential for including PDE in the NBS panel, early diagnosis and prompt precision treatment initiation.

Keywords: 2S,6S- and 2S,6R-oxopropylpiperidine-2-carboxylic acid; 6-oxopiperidine2-carboxylic acid; dried blood spot; expanded newborn screening; pyridoxine-dependent epilepsy; second-tier test
Corresponding author: Giancarlo la Marca, Newborn Screening, Clinical Biochemistry and Clinical Pharmacy Laboratory, Meyer Children’s Hospital IRCCS, 50139 Florence, Italy; and Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, 50139 Florence, Italy, E-mail:
Alessandra Bettiol and Giancarlo la Marca contributed equally to this work.

Funding source: European Union – Horizon 2020 research and innovation programme and Tuscany Region under the EJP RD COFUND

Award Identifier / Grant number: 825575

Acknowledgments

The research was conducted in the framework of the CHARLIE project (CHAnging Rare disorders of LysInE metabolism), funded by the European Joint Programme on Rare Diseases, 2020 call. We are grateful to the patients and families as well as our laboratory and clinical colleagues for their collaboration.

  1. Research ethics: We conducted all experiments in compliance with the Declaration of Helsinki (as revised in 2013) and with National and International guidelines for research studies on human subjects. The Institutional Review Board of Tuscany Region (Italy) approved this study (n.2021-11.02.2021).

  2. Informed consent: For these two patients with PDE, we obtained parents’ written authorization to use the residual neonatal DBS material. For the 5,405 and 35 neonatal DBS samples used for the first- and second-line tests, informed consent was not required as we treated all samples in a fully anonymous form and used as aggregated data.

  3. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: Not applicable.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: The research was conducted in the framework of the CHARLIE project (CHAnging Rare disorders of LysInE metabolism). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP No. 825575.

  7. Data availability: Raw data will be made available upon written reasonable request to the corresponding author.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-1230).

Received: 2024-10-22
Accepted: 2025-01-26
Published Online: 2025-02-04
Published in Print: 2025-06-26

© 2025 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2024-1230
Keywords for this article
2S,6S- and 2S,6R-oxopropylpiperidine-2-carboxylic acid; 6-oxopiperidine2-carboxylic acid; dried blood spot; expanded newborn screening; pyridoxine-dependent epilepsy; second-tier test

Articles in the same Issue

  1. Frontmatter
  2. Editorials
  3. The journey to pre-analytical quality
  4. Manual tilt tube method for prothrombin time: a commentary on contemporary relevance
  5. Reviews
  6. From errors to excellence: the pre-analytical journey to improved quality in diagnostics. A scoping review
  7. Advancements and challenges in high-sensitivity cardiac troponin assays: diagnostic, pathophysiological, and clinical perspectives
  8. Opinion Paper
  9. Is it feasible for European laboratories to use SI units in reporting results?
  10. Perspectives
  11. What does cancer screening have to do with tomato growing?
  12. Computer simulation approaches to evaluate the interaction between analytical performance characteristics and clinical (mis)classification: a complementary tool for setting indirect outcome-based analytical performance specifications
  13. Genetics and Molecular Diagnostics
  14. Artificial base mismatches-mediated PCR (ABM-PCR) for detecting clinically relevant single-base mutations
  15. Candidate Reference Measurement Procedures and Materials
  16. Antiphospholipid IgG Certified Reference Material ERM®-DA477/IFCC: a tool for aPL harmonization?
  17. General Clinical Chemistry and Laboratory Medicine
  18. External quality assessment of the manual tilt tube technique for prothrombin time testing: a report from the IFCC-SSC/ISTH Working Group on the Standardization of PT/INR
  19. Simple steps to achieve harmonisation and standardisation of dried blood spot phenylalanine measurements and facilitate consistent management of patients with phenylketonuria
  20. Inclusion of pyridoxine dependent epilepsy in expanded newborn screening programs by tandem mass spectrometry: set up of first and second tier tests
  21. Analytical performance evaluation and optimization of serum 25(OH)D LC-MS/MS measurement
  22. Towards routine high-throughput analysis of fecal bile acids: validation of an enzymatic cycling method for the quantification of total bile acids in human stool samples on fully automated clinical chemistry analyzers
  23. Analytical and clinical evaluations of Snibe Maglumi® S100B assay
  24. Prevalence and detection of citrate contamination in clinical laboratory
  25. Reference Values and Biological Variations
  26. Temporal dynamics in laboratory medicine: cosinor analysis and real-world data (RWD) approaches to population chronobiology
  27. Establishing sex- and age-related reference intervals of serum glial fibrillary acid protein measured by the fully automated lumipulse system
  28. Hematology and Coagulation
  29. Performance of the automated digital cell image analyzer UIMD PBIA in white blood cell classification: a comparative study with sysmex DI-60
  30. Cancer Diagnostics
  31. Flow-cytometric MRD detection in pediatric T-ALL: a multicenter AIEOP-BFM consensus-based guided standardized approach
  32. Impact of biological and genetic features of leukemic cells on the occurrence of “shark fins” in the WPC channel scattergrams of the Sysmex XN hematology analyzers in patients with chronic lymphocytic leukemia
  33. Assessing the clinical applicability of dimensionality reduction algorithms in flow cytometry for hematologic malignancies
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  35. Evaluation of sex-specific 0-h high-sensitivity cardiac troponin T thresholds for the risk stratification of non-ST-segment elevation myocardial infarction
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  40. Reply to “Is this quantitative test fit-for-purpose?”
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