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[15]
enzyme activities . For the screening of some diseases such as GALT deficiency and lysosomal disorders,
the enzyme activities may be measured by MS/MS, but the workflow must be adapted to the individual NBS
[16]
center . Moreover, in some disorders such as cystinuria and orotic aciduria, the specific biomarker is more
accurately analyzed in urine samples, sometimes used to complement DBS. This is further complicated in
X-linked conditions, such as Fabry disease, Hunter syndrome, and X-linked adrenoleukodystrophy , in
[17]
which the differentiation between an affected female carrier and a healthy individual may be challenging.

Adding a new disease that needs additional methodology to a national or regional NBS program not only
increases cost compared to using current methods, but also uses more of the already limited material of the
DBS sample. The actual NBS panorama in Europe is very heterogeneous , varying from country to country
[18]
and even within the same country, such as the case of Spain, where a minimum core of diseases to screen is
imposed by the Ministry of Health of the Central Government [19-21] . Still, the 17 autonomous regions, with
20 NBS centers, are free to develop further local implementations of NBS. This situation brings a problem of
inequity where, depending on the place of birth, one can have, for example, a positive detection of a fatty
acid oxidation disorder or not. Of course, there are justified reasons for the NBS heterogeneity among
different European regions depending on the varied frequency of some conditions, for example, in northern
countries compared to the Mediterranean ones, but at the same time, it may give rise to inequity of care and
may present difficulties with immigrants where it might not always be clear what NBS has been done in a
neonate [22-25] .

Although conventional NBS is a successful program, it has several limitations, some resulting from the lack
of a reliable neonatal biochemical marker, such as Wilson disease, while others being of organizational/
administrative, economic, or methodological reasons (e.g., homocysteine for classical homocystinuria is not
commonly measured as a first tier despite being the best marker). The goal of any NBS program is to deliver
rapid results to enable the initiation of a timely therapeutic strategy that would avoid the appearance of
irreversible disease complications. The availability of a rapid NBS result, indeed, depends on various
organizational factors, such as the established hours of life for the sample collection, the model of transport
to the reference NBS laboratory, and the coordination in the reference center until management by the
clinical metabolic team of the newborn tested positive is fully in place. The conventional NBS for metabolic
diseases, mainly of the intermediary metabolism (amino acids, organic acids, carbohydrates, and fatty
acids), was considered to require that the newborn had taken some food for at least 24 h to avoid false
negatives, but this consideration has been found not to be true for every biomarker (especially if ratios are
being used) [26,27] . Other known limitations of conventional NBS include preterm infants, parenteral
nutrition, transfusions, or metabolic decompensation due to various causes. In some centers, the collection
of DBS samples is performed in two steps, across two different days, and even including the collection of a
dried urine sample .
[28]

Since IMDs, by definition, have a genetic origin, there is an ongoing discussion to add other treatable IMDs
into NBS, using NGS as the screening test. Genomic testing as a first tier for IMDs has already been
introduced in the clinical setting for the rapid diagnosis of severe pediatric conditions in neonates and older
[29]
children in intensive care units , and some pilot studies are underway for the use of genome sequencing
techniques for NBS (gNBS) [30-32] . While gNBS may provide a more extensive disease identification , and
[33]
independence from the blood collection timing, there are also inherent specific challenges and
controversies, encompassing technical, interpretative, social, ethical, and economic aspects, as well as
implications at the healthcare level . Although well known in a diagnostic context (symptomatic
[34]
newborns), the analytical and clinical validity, sensitivity, and specificity of genome sequencing have not
been extensively examined in a screening context, mainly concerning healthy newborns . Furthermore,
[35]