Abstract
Objectives
Acylcarnitine and amino acid analyses of dried blood spot (DBS) samples using tandem mass spectrometry in newborn screening (NBS) programmes can generate false positive (FP) results. Therefore, implementation of second-tier tests (2TTs) using DBS samples has become increasingly important to avoid FPs. The most widely used 2TT metabolites include methylmalonic acid, 3-hydroxypropionic acid, methylcitric acid, and homocysteine.
Methods
We simultaneously measured 46 underivatised metabolites, including organic acids, acylglycine and acylcarnitine isomers, homocysteine, and orotic acid, in DBS samples using tandem mass spectrometry. To validate this method, we analysed samples from 147 healthy newborns, 160 patients with genetic disorders diagnosed via NBS, 20 patients with acquired vitamin B12 deficiency, 10 newborns receiving antibiotic treatment, and nine external quality control samples.
Results
The validation study revealed that 31 metabolites showed good analytical performance. Furthermore, this method detected key metabolites for all diseases associated with increased levels of the following acylcarnitines: C3, C4, C5, C4DC/C5OH, and C5DC. The sensitivity of this method to detect all diseases was 100 %, and the specificity was 74–99 %, except for glutaric aciduria type 1. This method can also be used to diagnose mitochondrial fatty acid β-oxidation disorders (FAODs) and urea cycle defects (UCDs).
Conclusions
We have described a 2TT panel of 31 metabolites in DBS samples based on an easy and rapid method without derivatisation. Its implementation allowed us to distinguish between different organic acidurias, some FAODs, and UCDs. This new strategy has increased the efficiency of our NBS programme by reducing FP and false negative results, second sample requests, and the time required for diagnosis.
Funding source: Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and the CERCA Programme/Generalitat de Catalunya
Funding source: Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), an initiative of the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, Spain)
Acknowledgments
Thanks are due to C. Martínez, G. Delgado, Y. Quintero, E. Ramon, N. Castillo, A. Muniente, and S. Richard, for the excellent technical assistance in the primary screening and helping in the second-tier analysis.
-
Research ethics: The local Institutional Review Board deemed the study exempt from review.
-
Informed consent: Informed consent was obtained from all individuals included in this study.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
-
Competing interests: The authors state no conflict of interest.
-
Research funding: This research was supported, in part, by Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), an initiative of the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, Spain), and by the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and the CERCA Programme/Generalitat de Catalunya.
References
1. Millington, DS, Kodo, N, Terada, N, Roe, D, Chace, DH. The analysis of diagnostic markers of genetic disorders in human blood and urine using tandem mass spectrometry with liquid secondary ion mass spectrometry. Int J Mass Spectrom Ion Process 1991;111:211–28. https://doi.org/10.1016/0168-1176(91)85056-r.Search in Google Scholar
2. Chace, DH, Adam, BW, Smith, SJ, Alexander, JR, Hillman, SL, Hannon, WH. Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays. Clin Chem 1999;45:1269–77. https://doi.org/10.1093/clinchem/45.8.1269.Search in Google Scholar
3. La Marca, G, Malvagia, S, Pasquini, E, Innocenti, M, Donati, MA, Zammarchi, E. Rapid 2nd-tier test for measurement of 3-OH-propionic and methylmalonic acids on dried blood spots: reducing the false-positive rate for propionylcarnitine during expanded newborn screening by liquid chromatography-tandem mass spectrometry. Clin Chem 2007;53:1364–9. https://doi.org/10.1373/clinchem.2007.087775.Search in Google Scholar PubMed
4. Turgeon, CT, Magera, MJ, Cuthbert, CD, Loken, PR, Gavrilov, DK, Tortorelli, S, et al.. Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry. Clin Chem 2010;56:1686–95. https://doi.org/10.1373/clinchem.2010.148957.Search in Google Scholar PubMed
5. Shigematsu, Y, Hata, I, Tajima, G. Useful second-tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria. J Inherit Metab Dis 2010;33(2 Suppl):S283–8. https://doi.org/10.1007/s10545-010-9111-9.Search in Google Scholar PubMed
6. Sarafoglou, K, Rodgers, J, Hietala, A, Matern, D, Bentler, K. Expanded newborn screening for detection of vitamin B12 deficiency. JAMA 2011;305:1198–200. https://doi.org/10.1001/jama.2011.310.Search in Google Scholar PubMed
7. Scolamiero, E, Villani, GR, Ingenito, L, Pecce, R, Albano, L, Caterino, M, et al.. Maternal vitamin B12 deficiency detected in expanded newborn screening. Clin Biochem 2014;47:312–17. https://doi.org/10.1016/j.clinbiochem.2014.08.020.Search in Google Scholar PubMed
8. Al-Dirbashi, OY, McIntosh, N, Chakraborty, P. Quantification of 2-methylcitric acid in dried blood spots improves newborn screening for propionic and methylmalonic acidemias. J Med Screen 2017;24:58–61. https://doi.org/10.1177/0969141316645824.Search in Google Scholar PubMed
9. Monostori, P, Klinke, G, Richter, S, Baráth, Á, Fingerhut, R, Baumgartner, MR, et al.. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders. PLoS One 2017;12:e0184897. https://doi.org/10.1371/journal.pone.0184897.Search in Google Scholar PubMed PubMed Central
10. Gramer, G, Fang-Hoffmann, J, Feyh, P, Klinke, G, Monostori, P, Mütze, U, et al.. Newborn screening for vitamin B12 deficiency in Germany—strategies, results, and public health implications. J Pediatr 2020;216:165–72.e4. https://doi.org/10.1016/j.jpeds.2019.07.052.Search in Google Scholar PubMed
11. Weiss, KJ, Röschinger, W, Blessing, H, Lotz-Havla, AS, Schiergens, KA, Maier, EM. Diagnostic challenges using a 2-tier strategy for methylmalonic acidurias: data from 1.2 million dried blood spots. Ann Nutr Metab 2020;7676:268–76. https://doi.org/10.1159/000508838.Search in Google Scholar PubMed
12. Pajares, S, Arranz, JA, Ormazabal, A, Del Toro, M, García-Cazorla, A, Navarro-Sastre, A, et al.. Implementation of second-tier tests in newborn screening for the detection of vitamin B-12 related acquired and genetic disorders: results on 258,637 newborns. Orphanet J Rare Dis 2021;16:195. https://doi.org/10.1186/s13023-021-01784-7.Search in Google Scholar PubMed PubMed Central
13. Hu, Z, Yang, J, Lin, Y, Wang, J, Hu, L, Zhang, C, et al.. Determination of methylmalonic acid, 2-methylcitric acid, and total homocysteine in dried blood spots by liquid chromatography–tandem mass spectrometry: a reliable follow-up method for propionylcarnitine-related disorders in newborn screening. J Med Screen 2021;28:93–9. https://doi.org/10.1177/0969141320937725.Search in Google Scholar PubMed
14. Dubland, JA, Rakić, B, Vallance, H, Sinclair, G. Analysis of 2-methylcitric acid, methylmalonic acid, and total homocysteine in dried blood spots by LC-MS/MS for application in the newborn screening laboratory: a dual derivatization approach. J Mass Spectrom Adv Clin Lab 2021;20:1–10. https://doi.org/10.1016/j.jmsacl.2021.03.001.Search in Google Scholar PubMed PubMed Central
15. Shigematsu, Y, Yuasa, M, Ishige, N, Nakajima, H, Tajima, G. Development of second-tier liquid chromatography-tandem mass spectrometry analysis for expanded newborn screening in Japan. Int J Neonatal Screen 2021;7:44. https://doi.org/10.3390/ijns7030044.Search in Google Scholar PubMed PubMed Central
16. Younesi, S, Yazdani, B, Taheri Amin, MM, Saadati, P, Jamali, S, Modarresi, MH, et al.. Incorporation of second-tier tests and secondary biomarkers to improve positive predictive value (PPV) rate in newborn metabolic screening program. J Clin Lab Anal 2022;36:e24471. https://doi.org/10.1002/jcla.24471.Search in Google Scholar PubMed PubMed Central
17. Ruoppolo, M, Malvagia, S, Boenzi, S, Carducci, C, Dionisi-Vici, C, Teofoli, F, et al.. Expanded newborn screening in Italy using tandem mass spectrometry: two years of national experience. Int J Neonatal Screen 2022;8:47. https://doi.org/10.3390/ijns8030047.Search in Google Scholar PubMed PubMed Central
18. Mak, J, Peng, G, Le, A, Gandotra, N, Enns, GM, Scharfe, C, et al.. Validation of a targeted metabolomics panel for improved second-tier newborn screening. J Inherit Metab Dis 2023;46:194–205. https://doi.org/10.1002/jimd.12591.Search in Google Scholar PubMed PubMed Central
19. Kilgore, MB, Platis, D, Lim, T, Isenberg, S, Pickens, CA, Cuthbert, C, et al.. Development of a universal second-tier newborn screening LC-MS/MS method for amino acids, lysophosphatidylcholines, and organic acids. Anal Chem 2023;95:3187–94. https://doi.org/10.1021/acs.analchem.2c03098.Search in Google Scholar PubMed PubMed Central
20. Forni, S, Fu, X, Palmer, SE, Sweetman, L. Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC-MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis. Mol Genet Metabol 2010;101:25–32. https://doi.org/10.1016/j.ymgme.2010.05.012.Search in Google Scholar PubMed
21. Cloppenborg, T, Janzen, N, Wagner, HJ, Steuerwald, U, Peter, M, Das, A. Application of a second-tier newborn screening assay for C5 isoforms. JIMD Rep 2014;13:23–6. https://doi.org/10.1007/8904_2013_275.Search in Google Scholar PubMed PubMed Central
22. Boemer, F, Schoos, R, de Halleux, V, Kalenga, M, Debray, FG. Surprising causes of C5-carnitine false positive results in newborn screening. Mol Genet Metabol 2014;111:52–4. https://doi.org/10.1016/j.ymgme.2013.11.005.Search in Google Scholar PubMed
23. Sinclair, GB, Ester, M, Horvath, G, Karnebeek, S, Stockler-Ipsirogu, S, Vallance, H. Integrated multianalyte second-tier testing for newborn screening for MSUD, IVA, and GAMT deficiencies. J Inborn Errors Metabol Screen 2016;4:1–7. https://doi.org/10.1177/2326409816666296.Search in Google Scholar
24. Poggiali, S, Ombrone, D, Forni, G, Malvagia, S, Funghini, S, Mura, M, et al.. Reducing the false-positive rate for isovalerylcarnitine in expanded newborn screening: the application of a second-tier test. J Inborn Errors Metabol Screen 2016;4:1–7. https://doi.org/10.1177/2326409816661355.Search in Google Scholar
25. Carling, RS, Burden, D, Hutton, I, Randle, R, John, K, Bonham, JR. Introduction of a simple second tier screening test for C5 isobars in dried blood spots: reducing the false positive rate for isovaleric acidaemia in expanded newborn screening. JIMD Rep 2018;38:75–80. https://doi.org/10.1007/8904_2017_33.Search in Google Scholar PubMed PubMed Central
26. Zhou, W, Cai, H, Li, H, Ji, Z, Gu, M. Quantification of differential metabolites in dried blood spots using second-tier testing for SCADD/IBDD disorders based on large-scale newborn screening in a Chinese population. Front Pediatr 2021;9:757424. https://doi.org/10.3389/fped.2021.757424.Search in Google Scholar PubMed PubMed Central
27. Adhikari, AN, Currier, RJ, Tang, H, Turgeon, CT, Nussbaum, RL, Srinivasan, R, et al.. Genomic analysis of historical cases with positive newborn screens for short-chain Acyl-CoA dehydrogenase deficiency shows that a validated second-tier biochemical test can replace future sequencing. Int J Neonatal Screen 2020;6:4.10.3390/ijns6020041Search in Google Scholar PubMed PubMed Central
28. Rebollido-Fernandez, MM, Castiñeiras, DE, Bóveda, MD, Couce, ML, Cocho, JA, Fraga, JM. Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism. Rapid Commun Mass Spectrom 2012;26:2131–44. https://doi.org/10.1002/rcm.6325.Search in Google Scholar PubMed
29. Fu, X, Xu, YK, Chan, P, Pattengale, PK. Simple, fast, and simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid using liquid chromatography and mass spectrometry (LC/MS/MS). JIMD Rep 2013;10:69–78. https://doi.org/10.1007/8904_2012_205.Search in Google Scholar PubMed PubMed Central
30. Gucciardi, A, Pirillo, P, Di Gangi, IM, Naturale, M, Giordano, G. A rapid UPLC-MS/MS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening. Anal Bioanal Chem 2012;404:741–51. https://doi.org/10.1007/s00216-012-6194-1.Search in Google Scholar PubMed
31. Václavík, J, Mádrová, L, Kouřil, Š, de Sousa, J, Brumarová, R, Janečková, H, et al.. A newborn screening approach to diagnose 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. JIMD Rep 2020;54:79–86. https://doi.org/10.1002/jmd2.12118.Search in Google Scholar PubMed PubMed Central
32. Pajares García, S, López Galera, RM, Marín Soria, JL, Argudo Ramírez, A, González de Aledo-Castillo, JM, Ribes Rubió, A, et al.. Impact of the inclusion of second-tier tests in the newborn screening program of Catalonia and in other international programs. Rev Esp Salud Pública 2020;94:e202012158.Search in Google Scholar
33. Matern, D, Tortorelli, S, Oglesbee, D, Gavrilov, D, Rinaldo, P. Reduction of the false-positive rate in newborn screening by implementation of MS/MS-based second-tier tests: the Mayo Clinic experience (2004–2007). J Inherit Metab Dis 2007;30:585–92. https://doi.org/10.1007/s10545-007-0691-y.Search in Google Scholar PubMed
34. Fisher, L, Davies, C, Al-Dirbashi, OY, Ten Brink, HJ, Chakraborty, P, Lepage, N. A novel method for quantitation of acylglycines in human dried blood spots by UPLC-tandem mass spectrometry. Clin Biochem 2018;54:131–8. https://doi.org/10.1016/j.clinbiochem.2018.01.020.Search in Google Scholar PubMed
35. Pickens, CA, Isenberg, SL, Cuthbert, C, Petritis, K. Combining first and second-tier newborn screening in a single assay using high-throughput chip-based capillary electrophoresis coupled to high-resolution mass spectrometry. Clin Chem 2021;67:1709–20. https://doi.org/10.1093/clinchem/hvab171.Search in Google Scholar PubMed
36. Oglesbee, D, Sanders, KA, Lacey, JM, Magera, MJ, Casetta, B, Strauss, KA, et al.. Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD). Clin Chem 2008;54:542–9. https://doi.org/10.1373/clinchem.2007.098434.Search in Google Scholar PubMed
37. Alodaib, A, Carpenter, K, Wiley, V, Sim, K, Christodoulou, J, Wilcken, B. An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples. Ann Clin Biochem 2011;48:468–70. https://doi.org/10.1258/acb.2011.010283.Search in Google Scholar PubMed
38. Janzen, N, Steuerwald, U, Sander, S, Terhardt, M, Peter, M, Sander, J. UPLC-MS/MS analysis of C5-acylcarnitines in dried blood spots. Clin Chim Acta 2013;421:41–5. https://doi.org/10.1016/j.cca.2013.03.001.Search in Google Scholar PubMed
39. Murko, S, Aseman, AD, Reinhardt, F, Gramer, G, Okun, JG, Mütze, U, et al.. Neonatal screening for isovaleric aciduria: reducing the increasingly high false-positive rate in Germany. JIMD Rep 2023;64:114–20. https://doi.org/10.1002/jmd2.12345.Search in Google Scholar PubMed PubMed Central
40. Lin, Y, Zheng, W, Chen, Y, Huang, C, Fu, Q, Chen, D, et al.. Incorporating second-tier genetic screening for multiple acyl-CoA dehydrogenase deficiency. Clin Chim Acta 2022;537:181–7. https://doi.org/10.1016/j.cca.2022.10.024.Search in Google Scholar PubMed
41. Shigematsu, Y, Yuasa, M, Hata, I, Nakajima, H, Tajima, G, Ishige, N, et al.. 2-Methylacetoacetylcarnitine in blood of beta-ketothiolase deficiency and HSD10 disease. Med Mass Spectrom 2019;3:43–7.Search in Google Scholar
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0216).
© 2023 Walter de Gruyter GmbH, Berlin/Boston
