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A novel fluorometric enzyme analysis method for Hunter syndrome using dried blood spots
Molecular Genetics and Metabolism 105 (2012) 519–521
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Brief Communication
A novel fluorometric 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 b
Division of Medical Genetics, Department of Pediatrics, Duke Medicine, Durham, NC, USA Advanced Liquid Logic, PO Box 14025, Research Triangle Park, NC, USA
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 microfluidics Microplate
a b s t r a c t Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate enzyme assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay compared well with a recently reported digital microfluidic 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 primarily affects males [1–3]. The disease is characterized by a deficiency of the lysosomal enzyme iduronate 2-sulfatase (IDS; 300823). This enzyme deficiency leads to a range of clinical presentations (mild to severe). For the most effective disease management of MPS II, including 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 interest in screening newborns for several LSDs where treatment is available, including MPS II. Digital microfluidics (DMF), a novel labon-a chip method, is a promising method for multiplexing fluorometric 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 fluorometry assay and to develop a Abbreviations: MPS II, (Mucopolysaccharidosis type II); DBS, (dried blood spots); LSD, (lysosomal storage diseases); DMF, (digital microfluidic); IDS, (iduronate-2sulfatase). ⁎ Corresponding author at: Biochemical Genetics Laboratory, Division of Medical Genetics, Department of Pediatrics, Duke Medicine, 801 Capitola Drive, Suite 6, Durham, NC 27713, USA. Fax: + 1 919 549 0709. E-mail address: [email protected] (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
technique that is more convenient and less invasive than the previously published method [6]. The new benchtop fluorometric assay and its validation are described, in addition to a comparison with the existing DMF assay. 2. Materials and methods Recombinant human α-L-iduronidase (>7.5 nmol/min/μg) was purchased from R&D Systems, Inc., (Minneapolis, MN), and 4methylumbelliferyl-α-L-iduronate-2-sulfate from Moscerdam Substrates (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 α-L-iduronidase (IDU) was prepared (10 μg/mL iduronidase in 0.05 mol/L sodium acetate, 1 mg/mL BSA, 0.01% Tween20 at pH 5.0) and stored in 10 μL aliquots at −80 °C. Likewise a stock solution of 4-methylumbelliferyl-α-L-iduronate-2-sulfate (4MU-IDUS) was prepared and stored in 15 μL 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-identified 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 establish the reference range for normal controls. De-identified MPS II-
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A.A. Tolun et al. / Molecular Genetics and Metabolism 105 (2012) 519–521
affected, untreated patient DBS samples (n = 16) were kindly provided by Advanced Liquid Logic (RTP, NC), the Biochemical Genetics Laboratory 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 Laboratory (Duke-BGL), which is certified by both College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendment (CLIA). Briefly, 3.2 mm punch from DBS card was extracted by gentle mixing in 100 μL of 0.1% Tween20 in microcentrifuge tube for 30 min at ambient temperature. 10 μL of the extracts were incubated with the 10 μL artificial substrate 4-methylumbelliferyl-α-L-iduronate-2sulfate disodium salt in the presence of α-L-iduronidase at acidic pH (5.0) for 20 h at 37 °C. The reaction was stopped by addition of 50 μL of 0.2 M sodium bicarbonate (pH 10.1) and microplates were read using a benchtop fluorometer (Tecan, Austria). The relative fluorescence 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 control DBS (n= 10) was assayed 5 times within one plate (intra-assay) and individually on 9 different days and plates (inter-assay). For stability 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 measurable range (0.2 μmol/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 Microfluidics (DMF) diagnostic platform has been described previously [4,5]. In brief, DMF used a lab-on-a-chip system where sub-microliter sized droplets and other liquid handling operations (dispensing, transportation and merging) were manipulated and controlled through computer software. IDS activity in each sample was measured using digital microfluidic 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-α-L-iduronate-2-sulfate, 1 μg/mL recombinant human α-L-iduronidase, 0.1 mol/L sodium acetate, 0.01 mol/L lead acetate, at pH 5.0. After incubation at 37 °C for 1 h, the reaction was terminated 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 fluorescence of 4-MU and the activity of IDS was expressed in micro moles of 4-MU product formed per hour per liter of blood.
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 fluorometric method.
when stored at − 20 °C and −80 °C (CV = 2.8–13%). Operator variation (%CV) was 15%. Fig. 2 shows the results of a comparison between the fluorometric 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 18 ± 12 μmol/L/h, (min–max: 9.0–39 μmol/L/h) and is broader than that of the microplate assay (21 ± 8.8 μmol/L/h, min– max: 12–33 μmol/L/h). Likewise, the MPS II-affected patient range derived from 6 DBS samples using the DMF assay measured higher (0.0–4.5 μmol/L/h versus 0.1–0.6 μmol/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
3. Results and discussion Fig. 1 shows the distribution of IDS activities obtained for the normal 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 (mean ± 2stdev) was 16 ± 7.6 μmol/L/h (min–max: 7.7–22 μmol/L/ h). The control range for the NBS samples was 21 ± 8.8 μmol/L/h (min–max: 12–33 μmol/L/h). The MPS II-affected patient range was 0.3 ± 0.5 μmol/L/h (min–max: 0.0–0.8 μmol/L/h). Known patients samples were clearly separated from unaffected control samples. The intraday and interday variations (%CV) of the fluorometric 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
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 microfluidic (ALL) fluorometric methods. Both methods clearly separated affected patient spots from the normal control samples.
A.A. Tolun et al. / Molecular Genetics and Metabolism 105 (2012) 519–521
assay methods accomplished clear and complete separation of affected patient and normal control ranges. The availability of a reliable microplate diagnostic assay that uses DBS offers significant advantages compared with the existing methods using blood plasma and cultured skin fibroblasts. Blood spot collection through heel or finger pricks is less invasive than skin biopsy for establishment of fibroblast 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 confirmation of diagnosis. 4. Conclusions We have developed and validated a single-step fluorometric microplate assay for IDS activity from DBS samples that is reproducible and robust. It represents a significant improvement over the previously reported MPS II enzyme assay method that required two separate incubation steps performed on cultured fibroblast cells. Conflict 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.
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Acknowledgments Partial funding for this work was provided by Shire Human Genetic 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). References [1] R. Martin, M. Beck, C. Eng, R. Giugliani, P. Harmatz, V. Munoz, J. Muenzer, Recognition and diagnosis of mucopolysaccharidosis II (Hunter syndrome), Pediatrics 121 (2008) e377–e386. [2] J. Muenzer, M. Beck, C.M. Eng, M.L. Escolar, R. Giugliani, N.H. Guffon, P. Harmatz, W. Kamin, C. Kampmann, S.T. Koseoglu, B. Link, R.A. Martin, D.W. Molter, M.V. Munoz Rojas, J.W. Ogilvie, R. Parini, U. Ramaswami, M. Scarpa, I.V. Schwartz, R.E. Wood, E. Wraith, Multidisciplinary management of Hunter syndrome, Pediatrics 124 (2009) e1228–e1239. [3] H. Zhou, P. Fernhoff, R.F. Vogt, Newborn bloodspot screening for lysosomal storage disorders, J. Pediatr. 159 (7–13) (2011) e11. [4] R.S. Sista, A.E. Eckhardt, T. Wang, C. Graham, J.L. Rouse, S.M. Norton, V. Srinivasan, M.G. Pollack, A.A. Tolun, D. Bali, D.S. Millington, V.K. Pamula, Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns, Clin. Chem. 57 (10) (2011) 1444–1451. [5] R. Sista, A.E. Eckhardt, T. Wang, M. Sellos-Moura, V.K. Pamula, Rapid, single-step assay for Hunter syndrome in dried blood spots using digital microfluidics, Clin. Chim. Acta 412 (2011) 1895–1897. [6] Y.V. Voznyi, J.L. Keulemans, O.P. van Diggelen, A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease), J. Inherit. Metab. Dis. 24 (2001) 675–680.
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Brief Communication
A novel fluorometric 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 b
Division of Medical Genetics, Department of Pediatrics, Duke Medicine, Durham, NC, USA Advanced Liquid Logic, PO Box 14025, Research Triangle Park, NC, USA
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 microfluidics Microplate
a b s t r a c t Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate enzyme assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay compared well with a recently reported digital microfluidic 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 primarily affects males [1–3]. The disease is characterized by a deficiency of the lysosomal enzyme iduronate 2-sulfatase (IDS; 300823). This enzyme deficiency leads to a range of clinical presentations (mild to severe). For the most effective disease management of MPS II, including 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 interest in screening newborns for several LSDs where treatment is available, including MPS II. Digital microfluidics (DMF), a novel labon-a chip method, is a promising method for multiplexing fluorometric 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 fluorometry assay and to develop a Abbreviations: MPS II, (Mucopolysaccharidosis type II); DBS, (dried blood spots); LSD, (lysosomal storage diseases); DMF, (digital microfluidic); IDS, (iduronate-2sulfatase). ⁎ Corresponding author at: Biochemical Genetics Laboratory, Division of Medical Genetics, Department of Pediatrics, Duke Medicine, 801 Capitola Drive, Suite 6, Durham, NC 27713, USA. Fax: + 1 919 549 0709. E-mail address: [email protected] (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
technique that is more convenient and less invasive than the previously published method [6]. The new benchtop fluorometric assay and its validation are described, in addition to a comparison with the existing DMF assay. 2. Materials and methods Recombinant human α-L-iduronidase (>7.5 nmol/min/μg) was purchased from R&D Systems, Inc., (Minneapolis, MN), and 4methylumbelliferyl-α-L-iduronate-2-sulfate from Moscerdam Substrates (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 α-L-iduronidase (IDU) was prepared (10 μg/mL iduronidase in 0.05 mol/L sodium acetate, 1 mg/mL BSA, 0.01% Tween20 at pH 5.0) and stored in 10 μL aliquots at −80 °C. Likewise a stock solution of 4-methylumbelliferyl-α-L-iduronate-2-sulfate (4MU-IDUS) was prepared and stored in 15 μL 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-identified 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 establish the reference range for normal controls. De-identified MPS II-
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A.A. Tolun et al. / Molecular Genetics and Metabolism 105 (2012) 519–521
affected, untreated patient DBS samples (n = 16) were kindly provided by Advanced Liquid Logic (RTP, NC), the Biochemical Genetics Laboratory 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 Laboratory (Duke-BGL), which is certified by both College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendment (CLIA). Briefly, 3.2 mm punch from DBS card was extracted by gentle mixing in 100 μL of 0.1% Tween20 in microcentrifuge tube for 30 min at ambient temperature. 10 μL of the extracts were incubated with the 10 μL artificial substrate 4-methylumbelliferyl-α-L-iduronate-2sulfate disodium salt in the presence of α-L-iduronidase at acidic pH (5.0) for 20 h at 37 °C. The reaction was stopped by addition of 50 μL of 0.2 M sodium bicarbonate (pH 10.1) and microplates were read using a benchtop fluorometer (Tecan, Austria). The relative fluorescence 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 control DBS (n= 10) was assayed 5 times within one plate (intra-assay) and individually on 9 different days and plates (inter-assay). For stability 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 measurable range (0.2 μmol/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 Microfluidics (DMF) diagnostic platform has been described previously [4,5]. In brief, DMF used a lab-on-a-chip system where sub-microliter sized droplets and other liquid handling operations (dispensing, transportation and merging) were manipulated and controlled through computer software. IDS activity in each sample was measured using digital microfluidic 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-α-L-iduronate-2-sulfate, 1 μg/mL recombinant human α-L-iduronidase, 0.1 mol/L sodium acetate, 0.01 mol/L lead acetate, at pH 5.0. After incubation at 37 °C for 1 h, the reaction was terminated 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 fluorescence of 4-MU and the activity of IDS was expressed in micro moles of 4-MU product formed per hour per liter of blood.
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 fluorometric method.
when stored at − 20 °C and −80 °C (CV = 2.8–13%). Operator variation (%CV) was 15%. Fig. 2 shows the results of a comparison between the fluorometric 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 18 ± 12 μmol/L/h, (min–max: 9.0–39 μmol/L/h) and is broader than that of the microplate assay (21 ± 8.8 μmol/L/h, min– max: 12–33 μmol/L/h). Likewise, the MPS II-affected patient range derived from 6 DBS samples using the DMF assay measured higher (0.0–4.5 μmol/L/h versus 0.1–0.6 μmol/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
3. Results and discussion Fig. 1 shows the distribution of IDS activities obtained for the normal 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 (mean ± 2stdev) was 16 ± 7.6 μmol/L/h (min–max: 7.7–22 μmol/L/ h). The control range for the NBS samples was 21 ± 8.8 μmol/L/h (min–max: 12–33 μmol/L/h). The MPS II-affected patient range was 0.3 ± 0.5 μmol/L/h (min–max: 0.0–0.8 μmol/L/h). Known patients samples were clearly separated from unaffected control samples. The intraday and interday variations (%CV) of the fluorometric 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
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 microfluidic (ALL) fluorometric methods. Both methods clearly separated affected patient spots from the normal control samples.
A.A. Tolun et al. / Molecular Genetics and Metabolism 105 (2012) 519–521
assay methods accomplished clear and complete separation of affected patient and normal control ranges. The availability of a reliable microplate diagnostic assay that uses DBS offers significant advantages compared with the existing methods using blood plasma and cultured skin fibroblasts. Blood spot collection through heel or finger pricks is less invasive than skin biopsy for establishment of fibroblast 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 confirmation of diagnosis. 4. Conclusions We have developed and validated a single-step fluorometric microplate assay for IDS activity from DBS samples that is reproducible and robust. It represents a significant improvement over the previously reported MPS II enzyme assay method that required two separate incubation steps performed on cultured fibroblast cells. Conflict 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.
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Acknowledgments Partial funding for this work was provided by Shire Human Genetic 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). References [1] R. Martin, M. Beck, C. Eng, R. Giugliani, P. Harmatz, V. Munoz, J. Muenzer, Recognition and diagnosis of mucopolysaccharidosis II (Hunter syndrome), Pediatrics 121 (2008) e377–e386. [2] J. Muenzer, M. Beck, C.M. Eng, M.L. Escolar, R. Giugliani, N.H. Guffon, P. Harmatz, W. Kamin, C. Kampmann, S.T. Koseoglu, B. Link, R.A. Martin, D.W. Molter, M.V. Munoz Rojas, J.W. Ogilvie, R. Parini, U. Ramaswami, M. Scarpa, I.V. Schwartz, R.E. Wood, E. Wraith, Multidisciplinary management of Hunter syndrome, Pediatrics 124 (2009) e1228–e1239. [3] H. Zhou, P. Fernhoff, R.F. Vogt, Newborn bloodspot screening for lysosomal storage disorders, J. Pediatr. 159 (7–13) (2011) e11. [4] R.S. Sista, A.E. Eckhardt, T. Wang, C. Graham, J.L. Rouse, S.M. Norton, V. Srinivasan, M.G. Pollack, A.A. Tolun, D. Bali, D.S. Millington, V.K. Pamula, Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns, Clin. Chem. 57 (10) (2011) 1444–1451. [5] R. Sista, A.E. Eckhardt, T. Wang, M. Sellos-Moura, V.K. Pamula, Rapid, single-step assay for Hunter syndrome in dried blood spots using digital microfluidics, Clin. Chim. Acta 412 (2011) 1895–1897. [6] Y.V. Voznyi, J.L. Keulemans, O.P. van Diggelen, A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease), J. Inherit. Metab. Dis. 24 (2001) 675–680.