Analysis of a second-tier test panel in dried blood spot samples using liquid chromatography-tandem mass spectrometry in Catalonia’s newborn screening programme

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.

An asymptomatic father diagnosed with 3-methylcrotonyl-CoA carboxylase deficiency following his son newborn screening test

“…This observation is particularly relevant to 3MCCD, which has become one of the most common IMDs diagnosed through NBS [ 2 ]. However, a number of unwanted false positive cases and/or heterozygous carriers are still identified warrating the implementation of expanded second-tier test panels [ 13 ]. In the pre-NBS time, 3MCCD was identified in children referred for various symptoms, including developmental delay, failure to thrive, hypotonia, seizures, cardiomyopathy, or metabolic disturbances such as hypoglycemia, hyperammonemia, ketoacidosis, or Reye syndrome [ 1 , [5] , [6] , [7] , 12 ].…”

Current Status of Newborn Bloodspot Screening Worldwide 2024: A Comprehensive Review of Recent Activities (2020–2023)

“…The economic value of NBS also has been reviewed along with a look at progress in the automated economic assessment of NBS [1022,1023]. In addition to a review of the value of genetic analyses for NBS confirmation [1024], current screening tools, and future screening perspectives [1025], a recent report describes the use of LC-MS/MS as a second-tier panel of 31 metabolites increasing the efficiency of NBS by reducing false-positive and false-negative results, second sample requests, and the time to diagnosis [1026]. A two-part report from the Ethics Commission of the Spanish Society for Human Genetics summarizes and addresses NBS and NGS issues moving forward [1027,1028].…”