Advances in Biological Chemistry, 2011, 1, 58-64
doi:10.4236/abc.2011.13008 Published Online November 2011 (
Published Online November 2011 in SciRes.
Reliability of enzyme assays in dried blood spots for diagnosis
of 4 lysosomal storage disorders
Romina Ceci, Pablo N. de Francesco, Juan M. Mucci, Lorena N. Cancelarich, Carlos A. Fossati,
Paula A. Rozenfeld*
LISIN, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
Email: *
Received 3 August, 2011; revised 12 September, 2011; revised 23 September, 2011.
Introduction: Lysosomal storage disord ers ( LSD ) a re
inherited diseases caused, in the majority of them, by
the deficiency of lysosomal enzymatic activities. Ob-
jectives: We aimed to analyze the usefulness of DBS
samples for diagnosis of 4 LSDs, with the availability
of a large quantity of patient samples. Design and
methods: Blood samples from previously diagnosed
patients with Fabry, Gaucher, Hunter, and Maro-
teaux-Lamy syndromes and normal control indi-
viduals, were collected and dispen-sed in filter paper,
and used for enzymatic activity determination. Re-
sults: Diagnosis of hemi/homo-zygous patients with
Fabry, Hunter and Maroteaux-Lamy diseases using
DBS samples showed ideal parameters of 100% sen-
sitivity and specificity. DBS assay for Gaucher dis-
ease would need a posterior confirmatory step. Con-
clusions: Leukocyte measu-rement is the only reli-
able way to diagnose Gaucher disease. For Hunter,
Fabry and Maroteaux-Lamy disorders discrimina-
tion between patients and controls seems adequate
by DBS.
Keywords: Lysosomal Storage Disorders; Diagnosis;
Dried Blood Spots; Sensitivity; Enzymatic Activity; Re-
Lysosomal storage disorders (LSD) are severe inherited
diseases caused, in the majority of them, by the defi-
ciency of lysosomal enzymatic activities, resulting in the
accumulation of specific substances, such as complex
lipids, glycoproteins and glycosaminoglycans. Clinical
manifestations are characterized by a spectrum of clini-
cal manifestations, which may include organ dysfunction,
neurological involvement and skeletal abnormalities [1].
Enzyme replacement therapy is now available for speci-
fictreatment of 6 LSD, and is on development or in use
in clinical trials for some others [2]. Early instauration of
therapy, before irreversible organ damage, is needed for
its better success. Therefore, early diagnosis is crucial.
Diagnosis process generally starts by clinical suspi-
cion by a physician [3]. However, due to age at onset of
signs and symptoms is variable, and clinical manifesta-
tions could be unspecific and multisystemic, LSD are
generally underdiagnosed and/or there could be a sub-
stantial delay between age at onset of manifestations and
age at diagnosis.
Diagnosis is confirmed by laboratory determination of
the specific enzymatic activity in leukocytes or culture
fibroblasts, using 4-methylumbelliferone conjugated ar-
tificial substrate. The use of leukocytes as the enzyme
source implies the isolation of these cells from a volume
(10 mL) of liquid blood, requires a homogenisation step
with a metal tip sonicator, and total protein measurement
[4]. On the other hand, the use of fibroblasts requires a
sample of skin biopsy, primary cell culture of skin fibro-
blasts (avoiding the high risk of contamination), and to
wait for the growing of fibroblasts to obtain enough cell
counts [5].
Measurement of lysosomal enzymes activities in dried
blood spots (DBS) on filter paper was introduced in
2001 [6] as a method with several advantages over test-
ing in leukocytes/fibroblasts. First, it requires only a few
drops of blood. Second, the main benefit is the possibili-
ty to mail the DBS samples to specialized laboratories,
making it possible to send samples to other cities, re-
gions or countries for testing. This is especially impor-
tant due to LSD laboratory diagnosis is currently under-
gone in specialized reference laboratories. Therefore
there are a few labs per country, or somewhere they are
missing. Finally, this method allows microplate adapta-
tion, permitting the simultaneously measurement of mul-
tiple samples and automation. Potentially, it could also
be applicable to newborn screening.
Due to different techniques of blood sample collection
could change the enzyme activity, specific lab recom-
R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64 59
mendations for collection should be follow [7], in terms
of the specific filter paper and use or not of anticoagu-
The usefulness of DBS for LSD diagnosis is not uni-
versally accepted by medical community. Generally,
physicians recommend assaying enzymatic activity in
leukocytes/fibroblasts in order to confirm an abnormal
result in DBS [8]. However, to our knowledge, there is
no study analyzing clinical parameters of efficacy of en-
zymatic activity determination on DBS for diagnosis of
the lysosomal storage disorders Fabry, Gaucher, Hunter
(Mucopolysaccharidosis II) and Maroteaux-Lamy (Mu-
copolysaccharidosis VI). For this reason, we aimed to
analyze the usefulness of DBS samples for diagnosis of
these 4 LSDs, with the availability of a large quantity of
patient samples.
2.1. Patients and Samples
Samples from previously diagnosed patients with Fabry
(n = 45), Gaucher (n = 30), Hunter (n = 85), and Maro-
teaux-Lamy(n = 22) diseases and normal control indi-
viduals (n = 155), were obtained for this study. All the
samples were taken before instauration of any specific
treatment. Samples from heterozygous Fabry (n = 59),
Hunter (n = 10) and Gaucher (n = 33) individuals were
also included. The protocol was approved by the scien-
tific committee of AADELFA according to provisions of
the Declaration of Helsinki in 1995. The nature and pur-
pose of the study and its possible risks were explained to
all volunteers. All the patients gave their informed con-
sent previous to the participation in the study.
Peripheral blood samples were collected by venopun-
cture, no anticoagulant was added. Immediatly, 50ul
drops were dispensed in circles of S & S nº903 filter
paper cards (Schleier and Schuell, Dassel, Germany).
The cards were allowed to dry for 4 hours at room tem-
In order to control sample quality a reference enzyme
was assayed in all DBS samples prior to the specific
enzyme test. The chosen reference enzyme was beta-
galactosidase (BGL1, EC, and was carried out
according to the method of Civallero et al. [9]. All the
samples showed normal values on BGL1 assay (data not
shown), confirming the good quality of the samples ana-
lyzed in this work.
2.2. Enzymatic Activity Determination
The determination of the enzymatic activities in DBS
samples specific for each of the 4 diseases were under-
gone by adaptation of reported methods (Table 1) stated
in Table 1 in samples from patients and control indi-
Alpha-galactosidase A (GLA): A 3 mm diameter cir-
cle from DBS filter paper was placed into a well of a
black microplate and 70 l of the reaction mixture con-
taining 3.57 mol/l 4-methylumbelliferyl-α-D-galactopy-
ranoside (Glycosynth, Cheshire, England) and 0.07 mol/l
N-acetylgalactosamine (Toronto Research Chemicals
Inc., North York, Ontario, Canada) in 0.15 mol/l acetate
buffer pH = 4.5 was added. After an incubation for 8 h at
37˚C, the stop solution (180 l of 1 mol/l ethylenediamine,
pH = 11.4) was added.
Beta-glucosidase (BGLU): A 3-mm diameter circle
from DBS filter paper was placed into a well of a black
microplate and 150 l of the reaction mixture containing
4-methylumbelliferyl-β-D-glucopyranoside 10 mmol/l (G-
lycosynth, Cheshire, England) and sodium taurocholate
(Sigma, Saint Louis, MO, USA) 50 mmol/l in 0.54 mol/l
phosphate-citrate buffer pH 5.5 was added. After an in-
cubation for 24 h at 37˚C, the stop solution (150 l of 0.5
mol/l glycine-NaOH buffer pH = 10.3) was added.
Iduronate-2-sulphatase (IDS): A 2-mm diameter circle
from DBS filter paper was placed into an eppendorf and
50 l BSA 0.2% was added to elute the blood. 10 l of
eluted blood in BSA was placed into a well of a black
microplate and continued as the method reported by
Vo z ny i et al. [11]. Briefly, 20 l of 4-Methylumbellifer-
-iduronato-2-sulfato 1.25 mM (Moscerdam substra-
tes, Oegstgeest, the Netherlands) in 0.1 M sodium ace-
tate pH = 5 containing 10mM lead acetate was added.
After an incubation for 24 h at 37˚C, 20 l of 0.05 M cit-
rate-phosphate pH = 4.5 buffer and 10 l of alfa-iduroni-
dase purified from bovine testis (Moscerdam substrates,
Oegstgeest, the Netherlands) were added. After an incu-
bation for 24 h at 37˚C, the stop solution (150 l of 0.5
mol/l sodium carbonate/bicarbonate pH = 10.7 buffer)
was added.
Table 1. List of the enzymes, abbrevations and bibliographic references of the enzyme assays methods.
Disease Enzyme Enzyme abbreviation Enzyme Classification number Reference
Fabry Alpha-galactosidase A GLA EC [6]
Gaucher Beta-D-glucosidase BGLU EC [10]
Hunter Iduronate-2-sulfatase IDS EC [11]
Maroteaux Lamy Arylsulfatase B ASB EC [8]
opyright © 2011 SciRes. ABC
R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64
Arylsulphatase B (ASB): A 2-mm diameter circle
from DBS filter paper was placed into a well of a black
microplate and 150 l of the reaction mixture containing
15 m mol/l lead acetate and 10 mmol/l 4-methylum-
belliferyl-sulfate (Glycosynth, Cheshire, England) in 15
mmol/l acetate buffer pH = 5.0. After incubation for 24 h
at 37˚C with agitation, the stop solution (150 ml of 0.085
mol/l glycine-NaOH buffer pH 10.5) was added.
In all cases, the fluorescence of the product (excitation
365 nm; emission 450 nm) was measured on a Twinkle
LB 970 fluorometer (Berthold Technologies, Bad Wild-
bad, Germany). A standard curve of 4-methylumbelli-
ferone (Sigma, Saint Louis, MO, USA) was used to ex-
trapolate fluorescence counts to moles of enzymatic
product. The enzymatic activity was expressed as mi-
cromoles of 4-methylumbelliferone produced per liter of
blood per hour.
2.3. Analitical Parameters
Sensitivity, specificity, positive (PPV) and negative
(NPV) predictive values and positive results proportion for
each of the 4 enzymatic methods for diagnosis of Fabry,
Gaucher, Hunter and Maroteaux-Lamy were calculated.
Each sample was assayed in duplicate. One blank was
included for each sample, consisted of addition of both
substrate and stop solutions at the same time at the end
of the incubation time. Appropriate incubation times for
each enzyme were chosen, among the linearity curve, in
order to obtain enough 4-MU to be detected by the
fluorometer and in comparison to the counts of the blank.
Intra-assay CVs were below 9% and inter-assay CVs
below 15% for all the enzymes.
Histogram of enzymatic activity of GLA, BGLU, IDS
and ASB from patients and normal control individuals is
shown in Figure 1. The analytical parameters calculated
for each of the methods is shown in Table 2.
3.1. GLA
Histogram of enzymatic activity of GLA from hemyzy-
gous, heterozygous and normal control individuals is
shown in Figure 1(a). Mean value of GLA from normal
controls was 6.12 ± 2.28 moles/l·h, and the cut-off
(mean - 2SD) was 1.56. As shown in Figure 1(a), there
is no overlap between the values from normal controls
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R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64 61
Figure 1. Histograms and individuals values, respectively, of enzymatic activity determination in DBS for diagnosis of Fabry (1(a)
and 1(b)), Gaucher (1(c) and 1(d)), Hunter (1(e) and 1(f)), and Maroteaux-Lamy (1(g) and 1(h)) diseases.
Tabl e 2 . Analytical parameters for the enzymatic activity de-
termination in DBS for diagnosis of Fabry, Gaucher, Hunter
and Maroteaux-Lamy diseases.
Fabry Gaucher Hunter Maroteaux Lamy
Sensitivity 100 97 100 100
Specificity 100 100 100 100
VPP 100 100 100 100
VPN 100 99 100 100
Positive results (%) 100 99 100 100
and male patients, giving a sensitivity and specificity of
100%. Moreover both predictive values are 100% (Table
2). Values from heterozygous females with Fabry disease,
showed a high variability, ranging from very low values
to normal ones.
3.2. BGLU
Histogram of values of BGLU enzymatic activity in pa-
tients and normal controls revealed a minimal (but pre-
sent) overlap between both groups (Figure 1(c)). Cut-
off value of BGLU enzymatic activity is 1.87 μmoles/l.h.
As can be seen in Figure 1(d), one confirmed Gaucher
patient exhibited an enzymatic activity value coincident
with the cut-off value of this enzymatic assay. This result
reduces the sensitivity of this method to 97%. However,
the specificity was 100%.
We carried out a parallel analysis of BGLU in leuko-
cytes and DBS of samples from patients belonging to the
3 groups. The results are shown in Figure 2. Samples
from a few normal controls and heterozygotes on DBS
revealed enzyme values close to or at the cut-off value;
however, the respective value in leukocyte is above its
cut-off. Moreover, a homozygous patient with a DBS
value close to cut-off, showed very low enzymatic activ-
ity in leukocyte homogenates.
3.3. IDS
IDS enzymatic activity values obtained from Hunter pa-
tients and normal individuals showed no overlap between
both groups, with a good separation (Figures 1(e) and (f)).
All analytical parameters of this method were 100%.
opyright © 2011 SciRes. ABC
R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64
Figure 2. Direct comparison of enzyme measurements from
DBS and leukocytes from Gacuher homozygotes, hetero-
zygotes and normal control patients.
Samples from heterozygous showed intermediate numbers.
3.4. ASB
Cut-off value as determined by this method was 2.28
moles/l.h. Enzymatic activity of ASB in DBS (Figures
1(g) and (h)) from all the homozygous patients with
MPS VI showed values below the cut-off. Analytical
parameters were 100%.
3.5. Stability of Samples at Room Temperature
Stability of the samples stored at room temperature
(20˚C - 25˚C) for different days was analyzed (Figure 3).
The observed enzyme activities of GLA, BGLU and IDS
show little changes, even at 60 days. Regarding ASB
activity, a small, but continuous reduction of enzyme
activity is revealed up to 60 days. However, the reduc-
tion is no more than 15%, and this reduction would no
modify the final diagnosis.
4. Discussion
After clinical suspicion of LSD enzymatic activity deter-
mination of lysosomal enzymes is traditionally under-
gonein leukocytes isolated from 10 ml of blood or fibro-
blasts culture. Diagnosis of lysosomal storage disorders in
DBS on filter paper was introduced in 2001 [6] as a
method with several advantages over testing in leuko-
cytes. One of the main advantages is the possibility to
mail the DBS samples to specialized laboratories by
ordinary mail at room temperature. The cost of shipment
Figure 3. Evaluation of stability of the samples stored at room temperature for different days. The result is ex-
pressed as the ratio (%) between the activity determined at each time point to reference activity (day 0).
opyright © 2011 SciRes. ABC
R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64 63
is rather low and accessible, and there is no need for
special conditions of temperature at transport, nor strict
limitations on time of shipment, nor need for customs
importation documents. Time and cost needed to per-
form an assay are important variables to take into ac-
count when a lab is going to implement a new technique
and when high amount of samples have to be processed.
The costs of each assay are directly dependent on the
cost of the specific reagent for each enzyme to be ana-
lyzed. However, there are no substantial differences in
the amount of substrate needed for DBS in comparison
to the determination in leukocytes. When using DBS,
you avoid the need for isolation and sonication of leu-
kocytes extracts. These processes could take 1 more
working days. Moreover, due to enzyme activity is ex-
pressed per liter of blood that is dependent on the di-
ameter of the spot; you do not have to determine protein
concentration. Assays in DBS reduce cost and time.
Substrates for GLA, BGLU and ASB are produced and
marketed by a few companies and IDS reagents are
manufactured by one company. Depending on the coun-
try, in order to acquire the substrates you could have a
distributor of the reagent, or, you have to import the re-
agents. In the case of import, procedures are dependent
on each country regulation. In summary, DBS technol-
ogy improves the accessibility of patients to accurate and
early diagnosis. Access to early diagnosis, at least at the
time of onset of clinical manifestations would improve
effectiveness of therapy. Specific enzyme replacement
therapy is available for each of the 4 diseases, whose
diagnosis in DBS is evaluated here.
Civallero et al. [9] evaluated twelve enzyme assays
for diagnosis of LSD, however they reported a need for
centrifugation before fluorescence measurement. In our
work, we incubate the reaction mixture and read the
fluorescence directly at the microplate, without cen-
trifugation. This means a simplification of the assay.
Measuring GLA activity in DBS samples resulted in a
test with 100% sensitivity and specificity for diagnosis of
male patients with Fabry disease. Female patients with
Fabry disease revealed variable values, most of them
above the cut-off level. Similar observations were re-
ported by Linthorst [12], and even when using leukocytes
[13]. It is a consequence of random X-inactivation process
[14] and not a problem of the method itself. Genetic
analysis should be always included to perform the con-
firmation of diagnosis of females with Fabry disease. This
DBS method enabled to carry out screening strategies in
at risk-populations involving large number of male sam-
ples [15,16], with the benefit to detect previously undiag-
nosed Fabry patients, and new Fabry families.
Development of BGLU enzymatic assay in DBS was
reported in 2002 [10] using a large cohort of Gaucher
patients. They did not find any overlap of values be-
tween patients and controls, however nor was cut-off nor
clinical efficacy parameters determined. We aimed to
evaluate these parameters in order to be aware of the
clinical feasibility of this assay. Based on our results,
determination of activity of BGLU in filter paper could
be useful as a screening method. Values close to or be-
low the cut off level should be confirmed by the deter-
mination of BGLU in leukocytes.
MPS diagnosis starts with clinical recognition of ty-
pical clinical manifestations such as coarse face, short
stature, dysostosis multiplex, hepatosplenomegaly, air-
way and heart disease. Different MPS types and sub-
types could share the phenotype, and also the type of
accumulated GAG [17], therefore demonstration of the
reduction of specific enzymatic activity is the confirma-
tory diagnosis. Various MPS enzymes could be assay in
DBS [8,9]. In this study we included the ones for MPS II
and VI. We found good efficacy of both methods. More-
over, we analyzed both enzymes (IDS and ASB) in all
MPS II and VI patients. It is important for differential
diagnosis with multiple sulfatase deficiency [18,19]. In
all the cases, the other sulphatase assayed was within the
range of normal controls (not shown).
Looking at the histograms obtained, the better separa-
tion between normal control values and hemy/homozy-
gous ones was observed for IDS activity, followed by
ASB, then GLA, and the poorest for BGLU. Diagnosis
of hemy/homozygous patients with Fabry, Hunter and
Maroteaux-Lamy diseases using DBS samples showed
ideal parameters of 100% sensitivity and specificity. In
conclusion, for these 3 disorders, these DBS methods are
reliable. Specific enzyme analysis in leukocytes of those
samples from patients who showed reduced values in
dried blood spots will confirm the diagnosis.
Stability of samples at room temperature is an impor-
tant issue, taking into account samples should be mailed
to the reference lab. Time process since obtaining the
sample to enzyme assay could be, sometimes, of around
30 days. Looking at the values of stability described here,
the result would be reliable, in accordance with other
authors [20]. We would recommend not extending the
analysis of a sample taken more than 30 days before,
especially for ASB activity.
Other methods using DBS have been developed for
LSD diagnosis. Immunoquantification of enzymes im-
plies the use of specific antibodies, and it is based on
quantification of the protein, nor the enzyme activity
[21]. However, a disadvantage of this method is that an-
tibodies could not be available for every lab. On the
other hand, MS/MS technology was also used for LSD
diagnosis in a convenient multiplex assay [22]; the ex-
pensive equipment could be an obstacle for labs from
opyright © 2011 SciRes. ABC
R. Ceci et al. / Advances in Biological Chemistry 1 (2011) 58-64
developing countries. Quality control materials to be
used for every method are being prepared [23], which
will be used to the development of a quality control sys-
tem to standardize the assays around the different refer-
ence laboratories.
In conclusion, in this work we analyzed the usefulness
of DBS samples for diagnosis of 4 LSDs. DBS methods
for IDS, ASB and GLA activities shown to be reliable,
however, BGLU DBS assay would need a posterior con-
firmatory step. We recommend evaluating available me-
thods for other lysosomal diseases diagnosis in DBS, in
order to evaluate its usefulness as screening and/or con-
firmatory methods.
Leukocyte measurement is the only reliable way to di-
agnose Gaucher disease. For Hunter, Fabry and Maro-
teaux-Lamy disorders discrimination between patients
and controls seems adequate by DBS.
This study was supported by grants from SHIRE HGT and Agencia
Nacional de Promoción Científicay Tecnológica.
[1] Scriver, C.R., Beaudet, A.L., Sly, W. S. and Valle, D.
(2001) Metabolic and molecular basis of inherited dis-
eases. 8th Edition, McGraw Hill, New York.
[2] Beck, M. (2010) Therapy for lysosomal storage disorders.
IUBMB Life, 62, 33-40.
[3] Wenger, D.A., Coppola, S. and Liu, S.L. (2003) Insights
into the diagnosis and treatment of lysosomal storage
diseases. Arch Neurol, 60, 322-328.
[4] Kolodny, E.H. and Mumford, R.A. (1976) Human leu-
kocyte acid hydrolases: Characterization of eleven ly-
sosomal enzymes and study of reaction conditions for
their automated analysis. Clinica Chimica Acta, 70, 247-
257. doi:10.1016/0009-8981(76)90426-5
[5] Coelho, J. and Giugliani, R. (2000) Fibroblasts of skin
fragments as a tool for the investigation of genetic dis-
eases: Technical recommendations. Genetics and Mo-
lecular Biology, 23, 269-271.
[6] Chamoles, N.A., Blanco, M. and Gaggioli, D. (2001).
Fab- ry disease: Enzymatic diagnosis in dried blood spots
on filter paper. Clinica Chimica Acta, 308, 195-196.
[7] Olivova, P., van der Veen, K., Cullen, E., et al. (2009)
Effect of sample collection on alpha-galactosidase a en-
zyme activity measurements in dried blood spots on filter
paper. Clinica Chimica Acta, 403, 159-162.
[8] Chamoles, N.A., Blanco, M.B., Gaggioli, D., et al. (2001)
Hurler-like phenotype: Enzymatic diagnosis in dried
blood spots on filter paper. Clinica Chimica Acta, 47,
[9] Civallero, G., Michelin, K., de Mari, J., et al. (2006)
Twelve different enzyme assays on dried-blood filter pa-
per samples for detection of patients with selected inher-
ited lysosomal storage diseases. Clinica Chimica Acta,
372, 98-102. doi:10.1016/j.cca.2006.03.029
[10] Chamoles, N.A., Blanco, M., Gaggioli, et al. (2002)
Gaucher and niemann-pick diseases enzymatic diagnosis
in dried blood spots on filter paper: retrospective diagno-
sis in newborn-screening cards. Clinica Chimica Acta,
317, 191-197. doi:10.1016/S0009-8981(01)00798-7
[11] Voznyi, Y.V., Keulemans, J.L.M., Beyer, E.M., et al.
(2001) A fluorogenic assay for the diagnosis of Hunter
disease (MPS II). Journal of Inherited Metabolic Disease,
24, 675-680.
[12] Linthorst, G., Vedder, A., Aerts, J., et al. (2005) Screen-
ing for fabry disease using whole blood spots fails to
identify one-third of female carriers. Clinica Chimica
Acta, 353, 201-203. doi:10.1016/j.cccn.2004.10.019
[13] Lukacs, Z., Hartung, R., Beck, M., et al. (2007) Direct
comparison of enzyme measurements from dried blood
and leukocytes from male and female fabry disease pa-
tients. Journal of Inherited Metabolic Disease, 30, 614.
[14] Lyon, M.F. (1961) Gene action in the X-chromosome of
the mouse (Mus musculus L.). Nature, 190, 372-373.
[15] Porsch, D.B., Nunes, A.C., Milani, V., et al. (2008) Fabry
disease in hemodialysis patients in southern Brazil:
prevalence study and clinical report. Renal Failure, 30,
825-830. doi:10.1080/08860220802353777
[16] Gaspar, P., Herrera, J., Rodrigues, D., et al. (2010) Fre-
quency of fabry disease in male and female haemodialy-
sis patients in Spain. BMC Medical Genetics, 11, 19.
[17] Neufeld, E.F. and Muenzer, J. (1995) The mucopolysac-
charidoses. In: Scriver, C.R., Beaudet, A.L., Sly, W.S.,
Valle, D., Eds., The Metabolic and Molecular Bases of
Inherited Disease, McGraw-Hill, New York, 2465.
[18] Cosma, M.P., Pepe, S., Annunziata, I., et al. (2003) The
multiple sulfatase deficiency gene encodes an essential
and limiting factor for the activity of sulfatases. Cell, 11 3,
445-456. doi:10.1016/S0092-8674(03)00348-9
[19] Dierks, T., Schmidt, B., Borissenko, L.V., et al. (2003)
Multiple sulfatase deficiency is caused by mutations in
the gene encoding the human C(alpha)-formylglycine
generating enzyme. Cell, 113, 435-444.
[20] Poeppl, A.G., Murray, G.J. and Medin, J.A. (2005) En-
hanced filter paper enzyme assay for high-throughput
population screening for fabry disease. Analysis Bio-
chemical, 337, 161-163. doi:10.1016/j.ab.2004.10.007
[21] Meikle, P.J., Grasby, D.J., Dean, C.J., et al. (2006) New-
born screening for lysosomal storage disorders. Mole-
cular Genetics and Metablism, 88, 307-314.
[22] Li, Y., Scott, C.R., Chamoles, N.A., et al. (2004) Direct
multiplex assay of lysosomal enzymes in dried blood
spots for newborn screening. Clinica Chimica Acta, 50,
1785-1796. doi:10.1373/clinchem.2004.035907
[23] De Jesus, V.R., Zhang, X.K., Keutzer, J, et al. (2009)
Development and evaluation of quality control dried
blood spot materials in newborn screening for lysosomal
storage disorders. Clinica Chimica Acta, 55, 158-164.
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