Multiplex Newborn Screening of Lysosomal Storage Diseases using Flow Injection Tandem Mass Spectrometry Mariana Barcenas 1 ; Martin Sadílek 1 , Susan Elliott 4 , František Tureček 1 ; C. Ronald Scott 2 ; Michael Gelb 1, 3 1 Department of Chemistry; 2 Department of Pediatrics; 3 Department of Biochemistry, University of Washington, Seattle, WA, USA, 4 Newborn Screening Washington State, Seattle, WA, USA Lysosomal Storage Disorders (LSD) comprise a group of over 40 degenerative diseases with a combined incidence of 1 in every 5,000 to 7,000. The incidence of LSDs as a group now competes with that of known disorders such as congenital hypothyroidism routinely screened for during the newborn period. For over a decade, our group has been developing enzyme assays based on tandem mass spectrometry for the detection of lysosomal storage disorders using Dried Blood Spots (DBS) in a format compatible with the workflow in newborn screening laboratories. Both flow-injection and high- performance liquid chromatography have been used to perform multiplex assays of several enzymes in a single DBS. We report on current efforts that are focused on developing a multiplex assay to screen 6 disorders (Krabbe, Gaucher, Niemann-Pick, Fabry, Pompe and Mucopolysaccharidosis I) using flow injection analysis. Introduction Acknowledgments •This project has been funded by NIH-NDDK, Grant R01 DK067859. •Waters Corporation, Dr. Michael Morris •Washington State Department of Health. References • Orsini, J.; Martin, M.; Showers, A.;Bodamer, O.; Zhang, X.; Gelb, M.; Caggana, M. Clin. Chim. Acta, 2012, 413, 1270-1273. • Spacil, Z., Tatipaka, H., Barcenas, M., Scott, C.R., Turecek, F., Gelb, M. Clin. Chem., 2013, in press • Blanchard, S.; Sadilek, M.; Scott, C.R.; Turecek, F.; Gelb, M. Clin. Chem. 2008, 54, 2067-2070. • Zhang, X.K.; Elbin, C.; Chuang, W.; Cooper, S.; Marashio, C.; Beauregard, C.; Keutzer, J. Clin. Chem. 2008, 54, 1725-1728. Conclusions • We have designed a multiplex assay protocol compatible with neonatal laboratories work flow with incubation in a common buffer and simple sample work up. • Critical parameters like source temperature and signal suppression were optimized. Other parameters were also investigated and optimized. • The method is ready for trials in neonatal laboratories and will be available commercially from Perkin Elmer in the near future. • Ongoing efforts are focused on expanding the capabilities of the multiplex assay to screen for nine disorders using Flow Injection (Mucopolysaccharidosis type II, IV-A and VI). Ethyl Acetate extraction DBS Overnight Incubation Reconstitute in 100 μL of 80:20 MeOH:H 2 O 5mM Ammonium Formate MS/MS Analysis 2min/sample Overview • A multiplex assay to screen for six lysosomal storage disorders was developed. • Sample workup was simplified and solid phase extraction proved to be unnecessary. • Quality Control Samples were used to test the multiplex assay. Enzyme Activity (μmol h -1 L -1 ) Abbreviation Lysosomal Enzyme Disorder GAA α‑glucosidase Pompe GLA α‑galactosidase Fabry GALC β‑galactocerebrosidase Krabbe ABG β‑glucocerebrosidase Gaucher ASM acid sphingomyelinase Niemann-Pick A/B IDUA α‑L‑iduronidase Mucopolysaccharidosis I In Source Substrate Decomposi2on as a Func2on of Source Temperature Methods Assay Protocol Blank QC Low QC Med QC High Healthy Adult DBS GAA 0.15 0.71 3.18 5.29 3.40 GLA 0.10 0.68 2.50 4.72 2.44 GALC 0.11 0.19 0.71 1.32 0.66 ABG 0.47 1.26 4.15 8.43 5.17 ASM 0.03 0.05 0.25 0.55 0.48 IDUA 0.42 6.24 23.67 47.92 35.40 Liquid/Liquid Extraction (LLE) vs Solid Phase Extraction (SPE) (Ethyl Acetate/Water) vs (Silica Gel Extraction) Enzyme Activity, μmol/L*h LLE SPE LLE SPE LLE SPE LLE SPE LLE SPE LLE SPE 0 5 10 15 20 25 GAA GLA GALC ABG ASM IDUA Analyte SRM transition (m/z) Cone(V) Collision Energy (eV) GAA‑S 660.35 → 560.30 18 15 GAA-P 498.30 → 398.24 18 15 GAA-IS 503.33 → 403.28 18 15 GLA‑S 646.33 → 546.28 18 15 GLA-P 484.28 → 384.23 18 15 GLA-IS 489.31 → 389.26 18 15 IDUA‑S 567.26 → 467.20 7 11 IDUA-P 391.19 → 291.13 7 11 IDUA-IS 377.17 → 277.12 7 11 ABG-S 644.50 → 264.20 22 21 ABG‑P 482.40 → 264.20 22 21 ABG‑IS 510.50 → 264.20 22 21 ASM‑S 563.40 → 184.00 15 22 ASM‑P 398.25 → 264.20 15 22 ASM‑IS 370.30 → 264.20 15 22 GALC‑S 588.50 → 264.20 16 20 GALC‑P 426.30 → 264.20 16 20 GALC‑IS 454.40 → 264.20 16 20 150 100 150 100 150 100 0.0 5.0×10 04 1.0×10 05 1.5×10 05 2.0×10 05 Source Temperature, ºC Product Signal Intensity ABG GALC GLA 150 100 150 100 150 100 0 1×10 03 2×10 03 3×10 03 4×10 03 Product Signal Intensity Source Temperature, ºC GAA IDUA ASM -90% -95% -78% -54% -40% -10% Signal Suppression by the Presence of Other Assay Compounds 3 mm DBS punch P P+IS mix P+IS+S mix P P+IS mix P+IS+S mix P P+IS mix P+IS+S mix P P+IS mix P+IS+S mix P P+IS mix P+IS+S mix P P+IS mix P+IS+S mix 0 2×10 04 4×10 04 1×10 06 2×10 06 3×10 06 Product Signal Intensity GAA GLA GALC ABG ASM IDUA