Multiplex Newborn Screening of Lysosomal Storage Diseases using Flow Injection Tandem Mass Spectrometry !!

Mariana Barcenas1; Martin Sadílek1, Susan Elliott4, František Tureček1; C. Ronald Scott2; Michael Gelb1, 3!1Department of Chemistry; 2Department of Pediatrics; 3Department 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:H2O!

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)!

Enz

yme

Act

ivity

, µm

ol/L

*h

LLESP

ELL

ESP

ELL

ESP

ELL

ESP

ELL

ESP

ELL

ESP

E0

5

10

15

20

25GAAGLAGALCABGASMIDUA

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

5.0×1004

1.0×1005

1.5×1005

2.0×1005

Source Temperature, ºC

Pro

du

ct S

ign

al I

nte

nsi

ty ABGGALCGLA

150

100

150

100

150

100

0

1×1003

2×1003

3×1003

4×1003

Pro

du

ct S

ign

al I

nte

nsi

ty

Source Temperature, ºC

GAAIDUAASM

-90%!

-95%!

-78%!

-54%!

-40%! -10%!

Signal Suppression by the Presence of Other Assay Compounds!

3 mm DBS punch!

PP

+IS

mix

P+I

S+S

mix P

P+I

S m

ixP

+IS

+S m

ix PP

+IS

mix

P+I

S+S

mix P

P+I

S m

ixP

+IS

+S m

ix PP

+IS

mix

P+I

S+S

mix P

P+I

S m

ixP

+IS

+S m

ix

0

2×10044×1004

1×1006

2×1006

3×1006

Pro

duct

Sig

nal I

nten

sity GAA

GLAGALCABGASMIDUA