Brief Communication

A novel uorometric enzyme analysis method for Hunter syndrome using dried
blood spots
Adviye A. Tolun
a, 1
, Carrie Graham
b, 1
, Qun Shi
a
, Ramakrishna S. Sista
b
, Tong Wang
b
, Allen E. Eckhardt
b
,
Vamsee K. Pamula
b
, David S. Millington
a
, Deeksha S. Bali
a,

a
Division of Medical Genetics, Department of Pediatrics, Duke Medicine, Durham, NC, USA
b
Advanced Liquid Logic, PO Box 14025, Research Triangle Park, NC, USA
a b s t r a c t a r t i c l e i n f o
Article history:
Received 10 November 2011
Received in revised form 13 December 2011
Accepted 13 December 2011
Available online 21 December 2011
Keywords:
Hunter syndrome
Enzyme assay
Dried blood spot
Iduronate-2-sulfatase
Digital microuidics
Microplate
Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by de-
ciency of iduronate-2-sulfatase (IDS). A convenient single-step uorometric microplate enzyme assay has
been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay com-
pared well with a recently reported digital microuidic method, from which it was adapted. Results show
that this DBS assay is robust and reproducible using both technologies.
2012 Elsevier Inc. All rights reserved.
1. Introduction
MPS II (Mucopolysaccharidosis type II) or Hunter syndrome
(#309900) is an X-linked lysosomal storage disorder (LSD) that pri-
marily affects males [13]. The disease is characterized by a deciency
of the lysosomal enzyme iduronate 2-sulfatase (IDS; 300823). This
enzyme deciency leads to a range of clinical presentations (mild to
severe). For the most effective disease management of MPS II, includ-
ing enzyme replacement therapy (ERT) or other treatment options, it
is important to diagnose affected patients as early as possible, before
tissue damage becomes irreversible. Hence, there has been recent in-
terest in screening newborns for several LSDs where treatment is
available, including MPS II. Digital microuidics (DMF), a novel lab-
on-a chip method, is a promising method for multiplexing uoromet-
ric enzymatic assays to screen for several LSDs simultaneously in
newborn blood spots. A detailed description of a novel single step
DMF assay for MPS II has been recently reported [4,5]. Our objective
was to modify already published DMF method for the diagnosis of
Hunter disease, using benchtop uorometry assay and to develop a
technique that is more convenient and less invasive than the previ-
ously published method [6]. The new benchtop uorometric assay
and its validation are described, in addition to a comparison with
the existing DMF assay.
2. Materials and methods
Recombinant human -L-iduronidase (>7.5 nmol/min/g) was
purchased from R&D Systems, Inc., (Minneapolis, MN), and 4-
methylumbelliferyl--L-iduronate-2-sulfate from Moscerdam Sub-
strates (Oegstgeest, Netherlands). 4-methylumbelliferone sodium
salt (4-MU), Tween20, sodium carbonate, molecular biology grade
bovine serum albumin (BSA) and all other reagents were from
Sigma Aldrich Corp (St. Louis, MO). Stock solution of recombinant
human -L-iduronidase (IDU) was prepared (10 g/mL iduronidase
in 0.05 mol/L sodium acetate, 1 mg/mL BSA, 0.01% Tween20 at pH
5.0) and stored in 10 L aliquots at 80 C. Likewise a stock solution
of 4-methylumbelliferyl--L-iduronate-2-sulfate (4MU-IDUS) was
prepared and stored in 15 L aliquots at 80 C (0.1 mol/L sodium
acetate, 0.01 mol/L lead acetate, 0.01% Tween20 at pH 5.0).
Iduronate-2-sulfatase (IDU-2S) assay solution was prepared fresh
daily and any unused solutions of IDU and 4MU-IDS were discarded.
Leftover de-identied blood spots belonging to diverse age groups
available through Duke Clinical Biochemical Genetics laboratory
(n=21) and newborn screening card samples provided by the
North Carolina Division of Public Health (n=77) were used to estab-
lish the reference range for normal controls. De-identied MPS II-
Molecular Genetics and Metabolism 105 (2012) 519521
Abbreviations: MPS II, (Mucopolysaccharidosis type II); DBS, (dried blood spots);
LSD, (lysosomal storage diseases); DMF, (digital microuidic); IDS, (iduronate-2-
sulfatase).
Corresponding author at: Biochemical Genetics Laboratory, Division of Medical Ge-
netics, Department of Pediatrics, Duke Medicine, 801 Capitola Drive, Suite 6, Durham,
NC 27713, USA. Fax: +1 919 549 0709.
E-mail address: bali0001@mc.duke.edu (D.S. Bali).
1
Both authors contributed equally to this work.
1096-7192/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2011.12.011
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism
j our nal homepage: www. el sevi er . com/ l ocat e/ ymgme
affected, untreated patient DBS samples (n=16) were kindly provid-
ed by Advanced Liquid Logic (RTP, NC), the Biochemical Genetics Lab-
oratory at Sir Ganga Ram Hospital, New Delhi, India, the Pediatric
Division, Cairo University, Cairo, Egypt, and from Medical Genetics
department, Porto Alegre, Brazil, and were used to set up the affected
patient range. The protocol was approved by the Duke IRB.
Clinical assays were carried out according to standard operating
procedures established in the Duke Clinical Biochemical Genetics Lab-
oratory (Duke-BGL), which is certied by both College of American
Pathologists (CAP) and Clinical Laboratory Improvement Amendment
(CLIA). Briey, 3.2 mm punch from DBS card was extracted by gentle
mixing in 100 L of 0.1% Tween20 in microcentrifuge tube for 30 min
at ambient temperature. 10 L of the extracts were incubated with
the 10 L articial substrate 4-methylumbelliferyl--L-iduronate-2-
sulfate disodium salt in the presence of -L-iduronidase at acidic pH
(5.0) for 20 h at 37 C. The reaction was stopped by addition of
50 L of 0.2 M sodium bicarbonate (pH 10.1) and microplates were
read using a benchtop uorometer (Tecan, Austria). The relative uo-
rescence values (RFU) were converted to enzyme activity units by
means of a 4-MU standard curve.
To establish intra-day and inter-day variability, a set of normal con-
trol DBS (n=10) was assayed 5 times within one plate (intra-assay)
and individually on 9 different days and plates (inter-assay). For stabil-
ity evaluation, DBS punches stored separately at ambient temperature,
4 C, 20 C and 80 C were analyzed at regular intervals. As part of
the validation process, operator variability was tested using the same
set of DBS punches analyzed by two different analysts in parallel. DBS
extracts were serially diluted (1:5, 1:10, 1:20, 1:50, 1:100 and 1:200)
and analyzed on the same plate to establish the lower end of measur-
able range (0.2 mol/L/h).
We further randomly selected 77 presumptively normal NBS
(newborn screening) spots and 6 MPS II-affected DBS spots available,
for a method comparison study between the benchtop microplate
and DMF methods.
Detailed analytical methodology for Digital Microuidics (DMF)
diagnostic platform has been described previously [4,5]. In brief,
DMF used a lab-on-a-chip systemwhere sub-microliter sized droplets
and other liquid handling operations (dispensing, transportation and
merging) were manipulated and controlled through computer soft-
ware. IDS activity in each sample was measured using digital microui-
dic cartridge by mixing one droplet (300 nL) of DBS extract with a
droplet of IDS substrate solution (300 nL); containing 1.125 mmol/L
4-methyl umbelliferyl--L-iduronate-2-sulfate, 1 g/mL recombinant
human -L-iduronidase, 0.1 mol/L sodiumacetate, 0.01 mol/L lead ace-
tate, at pH5.0. After incubation at 37 C for 1 h, the reaction was termi-
nated by mixing with a droplet (300 nL) of termination buffer at pH
10.0, containing; 0.2 mol/L sodium bicarbonate and 0.01% Tween 20.
Droplet with terminated reaction was transported to the detector to
measure the uorescence of 4-MUand the activity of IDS was expressed
in micro moles of 4-MU product formed per hour per liter of blood.
3. Results and discussion
Fig. 1 shows the distribution of IDS activities obtained for the nor-
mal laboratory controls (n=21), unaffected NBS spots (n=77) and
affected samples (n=16) using the new single-step microplate
assay. The enzyme activity range for the Duke-BGL control DBSs
(mean2stdev) was 167.6 mol/L/h (minmax: 7.722 mol/L/
h). The control range for the NBS samples was 218.8 mol/L/h
(minmax: 1233 mol/L/h). The MPS II-affected patient range was
0.30.5 mol/L/h (minmax: 0.00.8 mol/L/h). Known patients
samples were clearly separated from unaffected control samples.
The intraday and interday variations (%CV) of the uorometric
microplate assay were well within acceptable limits (10% and 9.5%,
respectively). IDS activity was stable for up to 1 month in DBSs stored
at ambient temperature and 4 C, and was stable for at least 2 months
when stored at 20 C and 80 C (CV=2.813%). Operator varia-
tion (%CV) was 15%.
Fig. 2 shows the results of a comparison between the uorometric
microplate MPS II assay performed in the Duke-BGL and the DMF
assay performed at Advanced Liquid Logic (ALL) using the same sets
of DBS samples. The control range for NBS specimens using the DMF
assay was 1812 mol/L/h, (minmax: 9.039 mol/L/h) and is
broader than that of the microplate assay (218.8 mol/L/h, min
max: 1233 mol/L/h). Likewise, the MPS II-affected patient range
derived from 6 DBS samples using the DMF assay measured higher
(0.04.5 mol/L/h versus 0.10.6 mol/L/h using the microplate
assay). These differences can be ascribed to procedural variations. In
particular, DMF employs a kinetic assay with an incubation time of
one hour that was designed as a screening method for NBS laboratory,
whereas the clinical method is an end-point assay. Nevertheless, both
Fig. 1. IDS activity levels measured in Duke clinical laboratory controls (n=21), known
MPS II-affected (n=16) and presumptive normal newborn (n=77) DBS specimens,
using a microplate uorometric method.
Fig. 2. Comparison of IDS enzyme activity levels obtained using the same normal
(n=77) and affected MPS II DBS (n=6) using microplate (Duke clinical laboratory)
and digital microuidic (ALL) uorometric methods. Both methods clearly separated
affected patient spots from the normal control samples.
520 A.A. Tolun et al. / Molecular Genetics and Metabolism 105 (2012) 519521
assay methods accomplished clear and complete separation of affect-
ed patient and normal control ranges.
The availability of a reliable microplate diagnostic assay that uses
DBS offers signicant advantages compared with the existing
methods using blood plasma and cultured skin broblasts. Blood
spot collection through heel or nger pricks is less invasive than
skin biopsy for establishment of broblast cultures or blood draw
for plasma isolation. Additionally, DBS samples collected from other
at-risk individuals can be conveniently mailed to distant reference
laboratories for quick non-invasive testing, and the leftover original
NBS specimen can be re-tested for conrmation of diagnosis.
4. Conclusions
We have developed and validated a single-step uorometric
microplate assay for IDS activity from DBS samples that is reproduc-
ible and robust. It represents a signicant improvement over the pre-
viously reported MPS II enzyme assay method that required two
separate incubation steps performed on cultured broblast cells.
Conict of interest
DSB and DSM have received research/grant support from Advanced
Liquid Logics for this study. CG, RSS, TW, AEE and VLP are currently
employed by Advanced Liquid Logics.
Acknowledgments
Partial funding for this work was provided by Shire Human Genet-
ic Therapies, Inc. (Lexington, MA) and Advanced Liquid Logics (RTP,
NC). We are grateful to Dr. Shu Chaing, Director, Newborn Screening
Laboratory, North Carolina Division of Public Health for providing
newborn dried blood spots (DBSs).
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