Environmental Engineering Reference
In-Depth Information
enzyme (involved in Fabry disease) being the most affected by age, only about
38 % of the median. The method based on API 3200 triple quadrupole instrument
mass spectrometer and multiple-reaction monitoring mode highlighted that affected
patients presented enzymatic activities corresponding to less than 20 % of the
age-matched controls.
The implementation of tandem mass spectrometry, based on substrate concen-
trations and enzyme activity measurements, into newborn screening programs
resulted in an exponential increase of the number of detectable metabolic disorders.
The synthesis of new reagents and substrates more closely related to homologous
with the natural enzyme precursors, improved the assay performance by enhancing
the mass spectrometry sensitivity and thus, decreasing the detection limit. More-
over, multiplex MS approaches have reduced the costs and time of analysis due to
the possibility of MS/MS method to detect multiple enzyme products simulta-
neously. Compared with standard methodologies, diagnosis by tandem mass spec-
trometry in dried blood spots exhibits considerable technical advantages.
Nowadays, it is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe,
and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by MS/MS.
8.5 Chip-Based Mass Spectrometry for Rapid Diagnosis
of Fabry Disease
In the last 5-6 years, different MS-based strategies
for monitoring the
α
-galactosidase A (GLA) enzyme in dried blood spots (DBS) [
58
,
61
-
63
], as well
as for measurement of the storage products in plasma [
64
] or urine have been
developed and implemented. Despite the facts that were capable to provide early
detection of LSDs in general and of Fabry disease in particular, false-negative
results [
65
] have been reported in a number of previous studies based on DBS
assays. In order to avoid the generation of false-negative or false-positive response,
it is necessary to ensure through the implemented methods a high level of repro-
ducibility and sensibility necessary for accurate comparative screening patient
vs. healthy control, as well as to avoid the carry-over from sample to sample
which might occur during successive ESI infusions. In 2013, a novel platform for
rapid and reliable diagnostic of Fabry disease, that eliminates all the drawbacks of
the DBS-MS method mentioned above, have been successfully implemented [
63
]
and it is based on enzyme assay and fully automated chip-nanoESI MS, collision-
induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS for a
total of 13 DBSs, 11 from healthy donors and 2 from Fabry patients.
The enzymatic assay workflow comprised five stages: (1) obtaining the DBSs by
uniform spotting 75
L of whole blood onto Whatman 903
®
specimen collection
paper, drying at room temperature and cutting with a 3 mm diameter puncher from
the middle of the circle, then storing them at 4
C; (2) enzymatic extraction from
μ
