Residual heel prick Dried Blood Spots (DBS) are valuable samples for retrospective investigation of inborn metabolic diseases (IMD) and biomarker analyses. Because many metabolites suffer time-dependent decay, we investigated the five-year stability of amino acids (AA) in residual heel prick DBS. In 2019/2020, we analyzed 23 AAs in 2170 residual heel prick DBS from the Dutch neonatal screening program, stored from 2013–2017 (one year at +4 °C and four years at room temperature), using liquid chromatography mass-spectrometry. Stability was assessed by AA changes over the five years. Hydroxyproline could not be measured accurately and was not further assessed. Concentrations of 19 out of the remaining 22 AAs degraded significantly, ranked from most to least stable: aspartate, isoleucine, proline, valine, leucine, tyrosine, alanine, phenylalanine, threonine, citrulline, glutamate, serine, ornithine, glycine, asparagine, lysine, taurine, tryptophan and glutamine. Arginine, histidine and methionine concentrations were below the limit of detection and were likely to have been degraded within the first year of storage. AAs in residual heel prick DBS stored at room temperature are subject to substantial degradation, which may cause incorrect interpretation of test results for retrospective biomarker studies and IMD diagnostics. Therefore, retrospective analysis of heel prick blood should be done in comparison to similarly stored heel prick blood from controls.

Homocystinuria (HCU) due to cystathionine-β-synthase deficiency is generally regarded as a rare disease, but within the Qatari population has an incidence of 1 in 1800 live births. Most newborn screening methods for HCU using dried blood spots (DBS) rely on the detection of an elevated methionine level or a rapid screen for total homocysteine (tHCY). However, screening based on methionine levels alone lacks specificity and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for tHCY exhibit variable results with high false positive rates. This report describes a LC-MS/MS method for detection of tHCY on DBS, with improved specificity. tHCY was extracted from DBS with a solution containing dithiothreitol and subsequently butylated with hydrochloric acid in n-butanol. The butyl esters were separated by liquid chromatography on a reverse-phase column and the homocysteine (HCY), detected by tandem mass spectrometry. The butyl ester of HCY eluted at 1.8 min. Total analysis time was 6.1 min per sample, including column flush and equilibration. This method allows for the quantification of tHCY over a linear range from 0.3 to 200 µM. Intraassay and interassay imprecision and recoveries were acceptable. Good concordance was observed with another LC-MS/MS method. Application of this method improves specificity and reduces false positive rates in screening for HCU.

With innovations in both the screening methodologies and treatment of diseases, newborn screening (NBS) programmes are confronted with an increasing number of candidate diseases

The assessment of newborn screening (NBS) algorithms’ performance to ensure quality improvements is a continuous process: false-positive referrals can enable optimisations in the shorter term, but false-negative referrals are often only discovered many years after the screening has taken place

Population-based newborn screening (NBS) using blood collected and dried on filter paper was developed in the 1960s and remains the international standard for NBS programs. Glue, used in the manufacture of dried blood collection cards, may present a source of contamination and is often considered as a possible cause of anomalous results in routine screening. Our study evaluates this potential contamination on NBS analyses. EBF#1003 glue was blotted onto dried blood collection cards made of Whatman grade 903 filter paper (Whatman#903) and adult whole blood was pipetted onto the dried glue blots. In addition, blank glue blots (i.e., no blood) and dried blood spots (DBSs) in the absence of glue were prepared. The DBSs and blank samples were run in duplicate as routine samples for NBS. DBS absorption time and diameter, the effect of glue on measurements (concentrations and variation) were assessed. DBS absorption time and shape were equivalent for DBSs prepared in the absence and presence of undiluted glue. DBS diameter increased when prepared in the presence of glue. When EBF#1003 was diluted prior to use, DBS absorption time increased, and DBSs were non-uniform. Glue, diluted or not, did not produce measurements above the established Limit of Detection (LOD) for all assays used in the current Dutch screening programme. For all analytes with concentrations in the quantifiable range, contamination with glue had no effect on measurement variation, as it appeared equivalent to variation in untreated DBSs. Our data show that, in the unlikely event of contamination of Whatman#903 with EBF#1003, there is no effect on the measured concentration of analytes.