Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry.
ABSTRACT Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories.
We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay.
In our study, the median enzyme activity measured in adults was generally increased 2-3-fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples.
The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.
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ABSTRACT: Persons with unexplained early-onset stroke have been targeted for screening surveys for Fabry disease, the most common of the three X-linked lysosomal disorders, because Fabry patients with stroke are more likely to have the life-threatening progressive cardiac and renal manifestations and would therefore most benefit from early diagnosis and intervention with enzyme replacement therapy (ERT). Among 175 Israeli patients with unexplained cryptogenic stroke screened for mutations in the Fabry α galactosidase A (GLA) gene, sequencing identified six with 2-4 GLA intronic variants, one of whose father and three sisters had the same variants. Two variants, c.640-16A>G (g.10115A>G) in intron 4 and c.1000-22C>T (g.10956C>T) in intron 6, were common to all patients. However, three males with a common four variant intronic haplotype had low residual enzyme activity and ~50% reduced mRNA expression. Transcript splice-site defects were not identified in any of the index cases and X-chromosome inactivation was not highly skewed in the six females. These data do not suggest that GLA intronic variants, per se, are pathogenic. Nonetheless, it is clear that a certain intronic haplotype in males with cryptogenic stroke is associated with reduced GLA expression and function.Gene 08/2014; · 2.20 Impact Factor
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ABSTRACT: Dried blood spots (DBS) typically consist in the deposition of small volumes of capillary blood onto dedicated paper cards. Comparatively to whole blood or plasma samples, their benefits rely in the fact that sample collection is easier and that logistic aspects related to sample storage and shipment can be relatively limited, respectively, without the need of a refrigerator or dry ice. Originally, this approach has been developed in the sixties to support the analysis of phenylalanine for the detection of phenylketonuria in newborns using bacterial inhibition test. In the nineties tandem mass spectrometry was established as the detection technique for phenylalanine and tyrosine. DBS became rapidly recognized for their clinical value: they were widely implemented in pediatric settings with mass spectrometric detection, and were closely associated to the debut of newborn screening (NBS) programs, as a part of public health policies. Since then, sample collection on paper cards has been explored with various analytical techniques in other areas more or less successfully regarding large-scale applications. Moreover, in the last 5 years a regain of interest for DBS was observed and originated from the bioanalytical community to support drug development (e.g., PK studies) or therapeutic drug monitoring mainly. Those recent applications were essentially driven by improved sensitivity of triple quadrupole mass spectrometers. This review presents an overall view of all instrumental and methodological developments for DBS analysis with mass spectrometric detection, with and without separation techniques. A general introduction to DBS will describe their advantages and historical aspects of their emergence. A second section will focus on blood collection, with a strong emphasis on specific parameters that can impact quantitative analysis, including chromatographic effects, hematocrit effects, blood effects, and analyte stability. A third part of the review is dedicated to sample preparation and will consider off-line and on-line extractions; in particular, instrumental designs that have been developed so far for DBS extraction will be detailed. Flow injection analysis and applications will be discussed in section IV. The application of surface analysis mass spectrometry (DESI, paper spray, DART, APTDCI, MALDI, LDTD-APCI, and ICP) to DBS is described in section V, while applications based on separation techniques (e.g., liquid or gas chromatography) are presented in section VI. To conclude this review, the current status of DBS analysis is summarized, and future perspectives are provided. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 9999:1–78, 2014Mass Spectrometry Reviews 09/2014; · 8.05 Impact Factor
Article: Fabry’s disease[Show abstract] [Hide abstract]
ABSTRACT: Fabry's disease is an X-linked lysosomal storage disorder caused by abnormalities in the GLA gene, which leads to a deficiency in α-galactosidase A. The abnormal accumulation of glycosphingolipids, primarily globotriaosylceramide, manifests as serious and progressive impairment of renal and cardiac function. In addition, patients experience pain, gastrointestinal disturbance, transient ischemic attacks and strokes. Disease presentation in female heterozygotes may be as severe as in males although women may also remain asymptomatic. This review covers all basic aspects of the disease such as epidemiology, pathophysiology, clinical presentation by systems, diagnosis, management, prevention, and repercussions on quality of life. With the development of enzyme replacement therapy in the past few years, early initiation of treatment was found to be key for reduction of disease burden in major affected organs with improvement in neuropathic pain, decreased cardiac mass and stabilization of renal function, gastrointestinal symptoms, and hearing. This review aims to raise the awareness of the signs and symptoms of Fabry disease as well as to provide guidelines for the diagnosis and treatment.Journal of the Neurological Sciences. 01/2014;
Multiplex Enzyme Assay Screening of
Dried Blood Spots for Lysosomal
Storage Disorders by Using Tandem
X. Kate Zhang,*Carole S. Elbin, Wei-Lien Chuang, Sa-
mantha K. Cooper, Carla A. Marashio, Christa Beaure-
gard, and Joan M. Keutzer
Genzyme Corporation, Framingham, MA; *address
Framingham, MA 01701. Fax 508-872-9080; e-mail
say screening for Pompe disease, Fabry disease, Gaucher
newborn screening. We modified the assay for high-
METHODS: We optimized enzyme reaction conditions
and procedures for the assay, including the concentra-
tions of substrate (S) and internal standard (IS), assay
cocktail compositions, sample clean-up procedures, and
mass spectrometer operation. The S and IS for each en-
zyme were premixed and bottled at an optimized molar
ratio to simplify assay cocktail preparation. Using the
new S:IS ratio, we validated the modified assay accord-
ing to CLSI guidelines. Stability of the S, IS, and assay
cocktails were investigated. Dried blood spots from 149
somal storage disorder (LSD) were tested using the
RESULTS: In our study, the median enzyme activity mea-
sured in adults was generally increased 2–3–fold com-
pared to the original method, results indicating higher
enzyme assays enabled unambiguous differentiation be-
CONCLUSIONS: The modified multiplex enzyme assay
with premixed S and IS is appropriate for use in high-
throughput screening laboratories.
Pick disease types A and B (NP A/B),1and Krabbe dis-
ciencies of acid ?-glucosidase (GAA, EC 126.96.36.199), acid
?-galactosidase (GLA, EC 188.8.131.52), acid ?-glucocere-
brosidase (GBA, EC 184.108.40.206), acid sphingomyelinase
220.127.116.11), respectively (1). The availability of disease-
specific therapies and the possibility that early diagnosis
may lead to improved patient outcomes have prompted
LSDs (2, 3). Li et al. (3) developed a novel approach to
tandem mass spectrometry (MS/MS). We optimized the
assay method reported by Li et al., modifying the proce-
dures for sample extraction, assay cocktail composition,
assay by using samples from healthy individuals (adult
The structures of the substrate (S) and internal
standard (IS) for GAA, GLA, GBA, ASM, and GALC
have been published (3). S and IS, individually and
mixed at a fixed S:IS ratio, were manufactured by
and 150, respectively. The sources of chemicals and ma-
terials are in the Data Supplement that accompanies the
online version of this Brief Communication at http://
Specimens from previously diagnosed patients
13 Gaucher disease, and 10 NP A/B patients) were col-
lected with the institutional research board–approved
protocol after patients gave written informed consent.
Specimens from 149 apparently healthy adults were
purchased from ProMedDx LLC. The preparation and
plement. One hundred deidentified newborn DBS
samples were obtained from the University Children’s
Hospital Vienna, Vienna, Austria.
During assay optimization, the S/IS stock solu-
viously reported (3). After optimization, we mixed the
S and IS for each enzyme in a fixed molar ratio to sim-
plify and standardize assay cocktail preparation. For
the GAA assay cocktail, 1.8 mL of 100 g/L 3-[(3-chol-
(CHAPS, CAS No. 75621-03-3) in water was added to
a vial of GAA S/IS and vortex-mixed briefly. Next
1Nonstandard abbreviations: NP A/B, Niemann-Pick disease types A and B; LSD,
lysosomal storage disorder; GAA, acid ?-glucosidase; GLA, acid ?-galactosi-
dase; GBA, acid ?-glucocerebrosidase; ASM, acid sphingomyelinase; GALC,
galactocerebrosidase; DBS, dried blood spots; MS/MS, tandem mass spectrom-
etry; S, substrate; IS, internal standard; CHAPS, 3-[(3-Cholamidopropyl)dimeth-
Clinical Chemistry 54:10
15.9 mL of 0.3 mol/L of phosphate citrate buffer and
0.3 mL of 0.8 mmol/L acarbose (Toronto Research
Chemicals) in water were added, and the vial was vor-
tex-mixed. The final GAA assay cocktail contained
0.67 mmol/L of GAA-S, 6.67 ?mol/L of GAA-IS, 10 g/L
The multiplex enzyme assay was adapted to a
96-well plate. Chloroform was replaced with ethyl ace-
tate. The composition and volume of solvents used in
liquid/liquid- and solid-phase extractions were modi-
GALC assay cocktail, thereby eliminating the enzyme
elution step. Complete details of the modified assay
protocol are in the online Data Supplement. We mon-
itored all analytes by using selected-reaction monitor-
ning (3), with an API 4000 triple-quadrupole MS/MS
(MDS Sciex). The enzyme activity of each sample was
calculated from the ion abundance ratio of product to
IS measured by MS. All enzyme activities were blank
suppression was effectively controlled, as demon-
strated by the regression analysis of calibration curves
collected during the validation study (Table 1 in the
online Data Supplement).
With use of the previously reported method (3),
ASM activities measured in samples from adults and
patients with NP A/B overlapped. Zn2?is required for
optimal activity of both secreted and lysosomal ASMs
(4–6), which are deficient in NP A/B patients. We
chose the 2 highest-activity NP A/B patient samples
and 3 low-activity adult samples and monitored their
activities with increasing concentrations of ZnCl2.
ASM activities in the adult samples increased with
ZnCl2, with maximal distinction between the normal
and disease samples at 0.5–1.5 nmol/L ZnCl2.
The previously reported GALC assay gave high
CVs attributable to low activity values, i.e., the mini-
mum activity in an adult sample was 0.6 ?mol/h/L in
samples. The S concentration was increased 5-fold to
plement). We determined that GALC activity was pre-
served by eluting samples directly into the GALC assay
of maltase glucoamylase, DBS can be used in the diag-
nosis of Pompe disease (3, 7). With the fluorogenic
substrate 4-methylumbelliferyl-?-glucoside, acarbose
concentrations of 3–9 ?mol/L are sufficient to inhibit
maltase glucoamylase activity in the assay (8, 9). We
found that with the synthetic substrate in this assay,
8 ?mol/L acarbose provided sufficient inhibition of
maltase glucoamylase and maintenance of GAA activ-
ity (Supplemental Fig. 2 in the online Data Supple-
ment). To minimize the effect of detergent on the
MS/MS, Triton X-100 was replaced by CHAPS, which
is more effectively removed by solid-phase extraction
and results in an increase in GAA activity.
tomation, the GLA assay was modified to use a 15-?L
instead of a 2.5-?L assay cocktail. Optimization of
N-acetyl galactosamine, buffer, and detergent concen-
trations increased GLA activity and decreased assay
variation. Changes to the GBA assay were limited to
increasing the S concentration. The modifications of
Table 1. Assay cocktail compositions used in the multiplex assay.
25 0.440.18 mol/L citrate-phosphate,
0.09 mol/L sodium acetate,
0.37 mol/L citrate-phosphate,
0.55 mol/L sodium acetate,
0.18 mol/L citrate-phosphate,
6 g/L CHAPS8 ?mol/L acarbose
25241.8 g/L sodium taurocholate96 mmol/L N-acetyl
25 0.489.6 g/L sodium taurocholate
25 0.240.6 g/L sodium taurocholate0.36 mmol/L zinc
1.2 g/L oleic acidGALC1b
3016.79.6 g/L sodium taurocholate
a10 of 70 ?L of DBS extractant was used in the assay, equivalent to 1/7 of 1 whole DBS.
bOne whole DBS was used in the assay.
Clinical Chemistry 54:10 (2008)
parison with the previous assay are summarized in
Supplemental Table 2 in the online Data Supplement.
The assay was validated according to the CLSI
plate imprecision was calculated using 20 punches
from 1 healthy adult sample and 1 corresponding pa-
in the GAA, GLA, GBA, ASM, and GALC assays were
for the disease sample were 10.1%, 24.8%, 24.8%,
23.1%, and 12.3%, respectively. Overall CVs in the
GAA, GLA, GBA, ASM, and GALC assays were 7.5%,
its of the blank for the GAA, GLA, GBA, ASM, and
h/L in whole blood, respectively. The limits of detec-
tion (defined as the 1.65 times the SD above the limits
of the blank) of the GAA, GLA, GBA, ASM, and GALC
whole blood, respectively. We verified that the GAA,
the range between the limits of detection and 7.2, 4.9,
5.3, 7.6 and 4.7 ?mol/h/L in whole blood, respectively.
Analysis performed with assay cocktails stored for 14
days at 37 °C, 30 days at 25 °C and 45 days at 2–8 °C
those for once-frozen assay solutions (Supplemental
Figs. 2–5 in the online Data Supplement). The use of
assay cocktails after 1, 2, and 3 freeze-thaw cycles re-
sulted in enzyme activities within 20% of those mea-
sured in freshly prepared assay solutions (Supplemen-
detectable MS/MS carryover in the assay.
Each of the 5 modified enzyme assays in the
multiplex format showed an unambiguous distinction
between samples from healthy individuals (adult and
newborn) and corresponding samples from patients
with LSDs (Fig. 1 and Supplemental Table 3 in the on-
line Data Supplement). The median activities in sam-
fold relative to the original method. In the GAA and
GALC assays, the separation between normal and dis-
ease samples became even more pronounced, and the
disease sample with the highest percentage of mean
15%, respectively. The limits of detection of each assay
were at least 2-fold below the maximum observed dis-
The availability of S and IS premixed for each en-
zyme assay has simplified cocktail preparation. The
modified assay proved to be reproducible and reliable
for the differentiation of normal and disease-specific
samples. Transfer of the high-throughput multiplex
enzyme assay to newborn screening laboratories should
be a straightforward process.
contributed to the intellectual content of this paper and
have met the following 3 requirements: (a) significant
data, or analysis and interpretation of data; (b) drafting
Fig. 1. Comparison of enzyme activities in DBS samples from healthy adults and newborns, patients with LSDs (with
corresponding enzyme deficiencies), and the remaining 4 disease samples in each enzyme assay.
NA, 149 normal adults; NB, 100 newborns; PD, 14 Pompe disease patients; GD, 13 Gaucher disease patients; NPD, 10
Niemann-Pick disease patients; FD, 13 Fabry disease patients; KD, 10 Krabbe disease patients; OL, patients with the other 4
LSDs; Wb, whole blood.
Clinical Chemistry 54:10 (2008)
approval of the published article.
Authors’ Disclosures of Potential Conflicts of Inter-
est: Upon submission, all authors completed the Disclo-
flicts of interest:
zyme; Joan M Keutzer, Genzyme.
Consultant or Advisory Role: None declared.
Stock Ownership: Christa Beauregard, Genzyme
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
role in the design of study, choice of enrolled patients,
review and interpretation of data, or preparation or
approval of manuscript.
physicians who contributed samples and to Dr. Olaf
Bodamer, University Children’s Hospital Vienna, Vi-
enna, Austria, for contributing newborn DBS for our
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