Clinical validation of the LC-MS/MS assay was performed retrospectively using 79 DBS samples from 32 different patients with PA or MMACBL from the NBS and Metabolic Centers in Heidelberg (Germany), Szeged (Hungary) and Zurich (Switzerland). In 14 patients, the newborn specimens prior to therapy initiation were also available (PA: n = 4; CblB n = 1; CblC n = 9); all other samples were obtained during therapy. Storage duration of the patient specimens before second-tier analysis in median (range) was 0.61 (0.01-8.90) years. For additional characterization of samples, we used the first–tier acylcarnitine profiles (electrospray ionization-MS/MS [18 (link)–20 ]) determined both at time of blood sampling at the respective site providing the specimen, as well as after storage (measured at the NBS Centers Heidelberg and Szeged). First–tier cutoff values for C3 and C3/C2 in the respective laboratories were the following: Heidelberg 5.5 μM and 0.22; Szeged: 5.75 μM and 0.26; and Zurich: 6.0 μM and 0.17. The 3OHPA, MMA and MCA levels were also measured in External Quality Assurance DBS samples for NBS (n = 7) from ERNDIM.
The validated assay is currently being evaluated prospectively in a pilot project for the extension of the German NBS panel named ‟Newborn screening 2020” at the NBS Center Heidelberg. In that prospective study, the reported LC-MS/MS assay is being applied as second-tier test using the same DBS specimen as in the primary screening.
All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. Written informed consent for regular newborn screening had been obtained from the parents of all individuals at time of blood sampling if required by national legislation. The anonymized, retrospective re-analysis of the specimens in the present study was approved by the Ethical Committees of the University of Szeged (217/2016-SZTE) and the University Children’s Hospital Zurich (2014-0211). At the University Hospital Heidelberg, parents of patients under treatment gave written informed consent for sample analysis for establishment of the second-tier strategies for the study NBS 2020. The study NBS 2020 was approved by the Ethical Committee of the University of Heidelberg (S-533/2015).
The validated assay is currently being evaluated prospectively in a pilot project for the extension of the German NBS panel named ‟Newborn screening 2020” at the NBS Center Heidelberg. In that prospective study, the reported LC-MS/MS assay is being applied as second-tier test using the same DBS specimen as in the primary screening.
All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. Written informed consent for regular newborn screening had been obtained from the parents of all individuals at time of blood sampling if required by national legislation. The anonymized, retrospective re-analysis of the specimens in the present study was approved by the Ethical Committees of the University of Szeged (217/2016-SZTE) and the University Children’s Hospital Zurich (2014-0211). At the University Hospital Heidelberg, parents of patients under treatment gave written informed consent for sample analysis for establishment of the second-tier strategies for the study NBS 2020. The study NBS 2020 was approved by the Ethical Committee of the University of Heidelberg (S-533/2015).
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