Dried blood spot screening

Detection of 3-O-methyldopa in dried blood spots for neonatal diagnosis of aromatic L-amino acid decarboxylase deficiency: The north-eastern Italian experience

Burlina A, et al. Mol Genet Metab. 2021;133:56–62.

Publication Date | March 2021
Authors | Burlina A, Giuliani A, Polo G, Gueraldi D, Gragnaniello V, Cazzorla C, Opladen T, Hoffmann G, Blau N, Burlina AP.
Citation | Mol Genet Metab. 2021;133:56–62.

https://pubmed.ncbi.nlm.nih.gov/33744095/

Patients with aromatic L-amino acid decarboxylase (AADC) deficiency present with a spectrum of phenotypic symptoms ranging from mild courses to a severe phenotype, the latter of which is observed in the majority of patients.¹ The diagnostic gap from first symptoms to the final diagnosis is often long, partly because the clinical symptoms of AADC deficiency are non-specific, and because diagnostic procedures are invasive, costly, time-consuming and limited to highly specialised laboratories.²

Currently, AADC deficiency diagnosis relies on molecular analysis of the DDC gene and neurotransmitter analysis of metabolites in cerebrospinal fluid (CSF), and/or AADC enzyme activity analysis.^1–3 Dry blood spot (DBS) testing has been identified by the International Working Group on Neurotransmitter Related Disorders as a screening method for AADC deficiency that should be investigated further.^1,3 It has been proposed to offer quantitative analysis of 3-O-methyldopa (3-OMD) in DBS with reduced measurement time and high specificity. Recently, this technique has been shown to have clinical validity as a reliable biomarker for neonatal diagnosis of AADC deficiency,^2,4 with immense clinical potential to improve and shorten the diagnostic work-up for AADC deficiency.⁵ It offers the advantages of being a simple, non-invasive and rapid method that has clinical relevance and utility in both high-risk and newborn screening (NBS), and could be an aid to helping more patients achieve a timely diagnosis and earlier treatment initiation.⁴

Until recently, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used to analyse 3-OMD concentrations in the DBS approach as part of an NBS programme in Taiwan⁴ and in a rapid high-throughput screening capacity in Germany.² While these have been shown to be sensitive techniques for quantifying 3-OMD in DBS samples, they rely on deuterated 3-OMD (3-OMD-d3) as the internal standard for MS/MS.^1,2,4 This can increase the time needed for the procedure and adds an extra layer of complexity to testing DBS samples, as additional chemicals need to be added to the kit reagents.¹

In this paper, Burlina et al describe a technique that streamlines the procedure for analysing 3-OMD levels in DBS samples using the labelled tyrosine internal standard already present amongst the reagents for NBS for tyrosinaemia. This offers two important advantages:

• The similar chemical structures of 3-OMD and tyrosine allows accurate quantification
• It avoids the need to add chemicals to the kit reagents, thereby preserving the validity of the kit certification¹

It is an efficient method that reduces the materials required for analysis and can allow (as this might vary from country to country) for screening of the entire NBS panel, plus 3-OMD using a single DBS.¹ In addition, it is rapid because 3-OMD does not require chromatographic separation. LC-MS/MS with 3-OMD-d3 is only used in the second-tier test to confirm positive screening results.¹

This approach was validated as part of an expanded access NBS programme in Padua, Italy. DBS samples were collected by the University Hospital of Padua from April to November 2020 – 21,867 DBS samples were collected on cards used for other NBS analyses (the 903™ sample collection card). 3-OMD concentration was calculated by comparing the analyte peak area to the ¹³C₆-tyrosine internal standard peak area because they have similar structures. DBS 3-OMD levels from a 25-year-old patient previously diagnosed with AADC deficiency were used for comparison. Although no patients with AADC deficiency were identified during the study period, the ¹³C₆-tyrosine internal standard for MS/MS detection of 3-OMD levels in DBS samples was validated.¹

3-OMD in DBS samples from 66 newborns (>7 days of life) and 41 children (>1 year old) in the general population were also measured, between March and April 2020, as part of the validation for quantification of 3-OMD. The concentration of 3-OMD in healthy controls was negatively correlated with age, and the reference interval of 3-OMD was defined as the value range between the first and 99th percentiles of the control groups. The concentration reference ranges of 3-OMD with respect to age are shown in Table 1.

For residual neonatal DBS specimens with first-tier 3-OMD concentrations above the referral cut-off, measurements of 3-OMD-d3 for second-tier testing by LC-MS/MS were measured.¹ This provisional cut-off for the pre-pilot study and the first 6 months of NBS was set at the 99th percentile in 2,370 newborns (48–72 hours of life), corresponding to 3.4 μmol/L.¹ The second-tier test cut-off was determined by analysing 102 DBS samples collected from healthy newborns (positive if 3-OMD >0.91 μmol/L).¹

Currently, the test is included as part of the expanded NBS program for north-eastern regions of Italy and is gathering further data to better define the normal reference range. To date, 21,867 DBS samples from newborns have been screened, but no affected patients have been identified. One false positive was noted and was associated with maternal L-Dopa therapy.¹

The diagnosis of AADC deficiency is often a difficult and time-consuming process. Furthermore, the non-specific symptoms and the rarity of the condition make diagnosis especially challenging for clinicians. The inclusion of 3-OMD testing to NBS programmes is feasible and beneficial. The method presented by Burlina et al can be incorporated into existing systems with relative ease as it involves currently used reagents and procedures. Although not wholly diagnostic, raised levels of 3-OMD may help inform physicians whether to proceed with a full diagnostic work-up.