Neonatal Screening for Lysosomal Storage Disorders (LSD) by Tandem Mass Spectrometry (MSMS)

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1 Neonatal Screening for Lysosomal Storage isorders (LS) by Tandem Mass Spectrometry (MSMS) Enzo Ranieri and Samantha Stark 1 Head, Neonatal Screening Centre & Biochemical Genetics (G&MP) irectorate of Genetics and Molecular Pathology Women s and Children s Hospital Campus, Adelaide SA Pathology, South Australia, Australia enzo.ranieri@health.sa.gov.au University of Adelaide, epartment of Pediatrics South Australia, Australia enzo.ranieri@adelaide.edu.au 1 Head, National Referral Laboratory (G&MP)

2 Women s and Children s Hospital Adelaide, South Australia Summary Statistics 2008/2009 Budget 36.5 million Emergency attendances 55,502 - Women 20,850 - Children 35,652 Admissions 41,595 - Women 19,480 - Children 22,115 ept. Biochemical Genetics Births 5,895 Beds Women Children ICU/SC 54 Average bed ccupancy 91.5% SAPathology at the Women s & Children s Hospital

3 SAPathology at the Women s & Children s Hospital irectorate of Genetics and Molecular Pathology 168 Tenured and 86 Research funded Staff Tertiary Paediatric & bstetric/gynaecological teaching Hospital affiliations with the University of Adelaide epartments of Paediatrics and bstetric & Gynaecology. epartments Clinical Genetics Molecular Genetics Biochemical Genetics Clinical Genetics Cytogenetics & Cancer Paediatric Biochemical Genetics Metabolic Clinic National Referral Laboratory Metabolic Laboratory Antenatal Screening Neonatal Screening

4 Screening & iagnosis of LS on ried Blood-spots Undertaken a study to investigate the use of the PE-LS 6-Plex MSMS substrates for NBS GAUCHER- β-glucocerebrosidase (ABG) KRABBE- Galactocerebrosidase β-galactosidase (GALC) NIEMAN-PICK A/B - Sphingomyelinase (ASM) FABRY- α-galactosidase (GLA) PMPE- α-glucosidase (GAA) MPS I- Mucopolysaccharidosis type I Iduronidase (IUA) Perform analysis on a Sciex MSMS API5000 Analysis method by Flow injection (FIA) & LC-MSMS etermine enzyme activity from dried blood-spots in:- Normal newborn population Confirmed true positive (CTP) cases

5 Relative incidence of LS in Australia 1 in 7,000 Based on Australian diagnoses ( ) Meikle et al JAMA

6 Substrates Stored in Lysosomal Storage isorders Wolman I-cell Sandhoff GM1- gangliosidosis Tay-Sachs (1) Cystinosis Salla Pompe's thers (n=18) MPS I MPS II MPS IIIA Glycosaminoglycan (sulphated saccharides) Sphingosine analogues Glycogen Amino acid Monosaccharide Niemann-Pick (A and B) Krabbe MPS IVA thers Niemann-Pick type C MPS IIIB MPS VI Metachomatic Leukodystrophy MPS III Fabry Gaucher Based on Australian diagnoses ( )

7 Lysosomal Storage isorders (LS) Cultured skin fibroblasts from normal & an LS affected. 5µm 5µm Control LS affected

8 PE Six-Plex FIA-MSMS substrates & internal stable isotopes. GAUCHER β-glucocerebrosidase (ABG) 3 C H NH H (CH 2 ) 8 ABG - IS g/mol NIEMANN-PICK A/B Sphingomyelinase (ASM) 2 C C 2 H NH H ASM - IS g/mol 2 C C C 3 2 (CH 2 ) 12 CH 3 KRABBE Galactocerebrosidase β-galactosidase (GALC) 3 C H C 2 H NH H GALC - IS g/mol FABRY α-galactosidase (GLA) t-bu H N N H N (CH 2 ) 6 GLA - IS g/mol (CH 2 ) 12 CH 3

9 PE Six-Plex FIA-MSMS substrates & internal stable isotopes. PMPE α-glucosidase (GAA)) H t-bu H N N H N (CH 2 ) 7 GAA - IS g/mol MPS I Mucopolysaccharidosis type I (IUA) H H N N (CH 2 ) 4 H N MPS - I - IS g/mol

10 MRM pairs for each LS substrate and product MRM Pairs Analyte Q1 Mass Q2 Mass ABG-S ABG-IS ABG-P ASM-S ASM-IS ASM-P GAA-S GAA-IS GAA-P MRM Pairs Analyte Q1 Mass Q2 Mass GALC-S GALC-IS GALC-P GLA-S GLA-IS GLA-P IUA-S IUA-IS IUA-P

11 FIA MSMS 1. Addition 30µL of reagent to each well in a microtitre plate. 2. Incubate at 37 o C for 18h and shake at 400rpm. 3. Add 100µL methanol:ethyl-acetate (MeH:EA, 50:50) to quench followed by centrifugation 4. Transfer to deep-well microtitre plates and add 400µL of EA & 200µL water 5. Centrifuge for 5 minutes to separate layers. 6. Transfer 75µL of the top layer to a microtitre plate 7. Evaporate at room temperature under a nitrogen stream 8. Reconstitute in 150µL solvent flow buffer 9. Mix at 400rpm and analyse on API5000 MSMS API5000 MSMS

12 FIA of LS Substrates, Products & Stable Isotopes XIC of +MRM (18 pairs (Turbo Spray) Max. 1.8e6 cps. 6.0e4 Intensity, cps 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min rder of XIC in the TIC 1. ASM-S 2. GAA-S 3. GLA-S 4. ABG-S 5. GAA-S 6. GAA-IS 7. ABG-IS 8. GLA-IS 9. ASM-IS 10. GALC-IS 11. IUA-S 12. IUA-IS 13. ABG-P 14. ASM-P 15. GALC-P 16. GLA-P

13 Analytical Performance Normal dried blood spot- 10 repeated estimates (3mm) Blood-Spot Stats ABG ASM GAA GALC GLA IUA Normal A Mean CV% Normal B Mean CV% Normal C Mean CV% Normal Mean CV%

14 Blood-spot Normal vs Affected Gaucher (ABG) Pompe (GAA) ABG Enzyme Activity GAA Enzyme Activity Normal Gaucher (592) (20) Normal Pompe (592) (20)

15 Blood-spot Normal vs Affected Niemann-Pick A/B (ASM) MPS-I (IUA) ASM Enzyme Activity IUA Enzyme Activity Normal NP-A/B (592) (1) Normal MPS-I (592) (6)

16 Blood-spot Normal vs Affected Krabbe (GALC) Fabry (GLA) GALC Enzyme Activity GLA Enzyme Activity Normal Krabbe (592) (5) 0 Normal Fabry Fabry-Hets (592) (14) (28)

17 Blood-spot Normal vs Affected Krabbe (GALC) Fabry (GLA) GALC Enzyme Activity GLA Enzyme Activity Normal Krabbe (592) (5) 0 Normal Fabry Fabry-Hets (592) (14) (28)

18 LC-MSMS for Confirmatory and iagnostic eveloped an n-line LC-MSMS method C18 column (100mm) on a Sciex API5000 MSMS instrument Gradient of: buffer A; Water + 0.1% Formic acid & buffer B; 50:50 Acetonitrile/Methanoi + 0.1% Formic acid Perform analysis on a Sciex MSMS API5000 Analysis method by Flow injection (FIA) & LC-MSMS etermine enzyme activity from dried blood-spots in:- Normal newborn population Confirmed true positive (CTP) cases

19 LC-MSMS for IUA MPS I- α Iduronidase Intenity, cps XIC of +MRM (19 pairs): / a I: - IUA IS from Sample 2 (mix) of e.wiff (Turbo Spray) Max. 1.4e6 cps. 5.3e6 Step Time(min) µl/min A (%) B (%) 5.0e e e6 3.5e6 3.0e6 2.5e6 2.0e6 Buffer A: Water, 0.1% formic Buffer B: 50% ACN, 50% MeH, 0.1% formic Red= substrate Blue= internal std 1.5e e6 5.0e Time, min

20 IUA MPS I- α Iduronidase Normal vs Affected

21 LC-MSMS for KRABBE- Galactocerebrosidase β-galactosidase (GALC) XIC of +MRM (19 pairs): / a I: - GALC IS from Sample 2 (mix) of e.wiff (Turbo Spray) Intenity, cps 5.5e6 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 Step Time(min) µl/min A (%) B (%) Buffer A: Water, 0.1% formic Buffer B: 50% ACN, 50% MeH, 0.1% formic Red= substrate Blue= internal std Max. 1.9e6 cps. 2.0e e6 1.0e6 5.0e Time, min

22 KRABBE- GALC activity Normal/Affected/Hets/Psuedo

23 LC-MSMS for FABRY α-galactosidase (GLA) 5.3e6 XIC of +MRM (19 pairs): / a - I: GLA IS from Sample 2 (mix) of e.wiff (Turbo Spray) Max. 3.1e6 cps. Intensity, cps 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e Step Time(min) µl/min A (%) B (%) Buffer A: Water, 0.1% formic Buffer B: 50% ACN, 50% MeH, 0.1% formic Red= substrate Blue= internal std 1.0e6 5.0e Time, min

24 GLA FABRY α-galactosidase Normal/Affected & Heterozyogote

25 Summary from the Adelaide Study The PE-6-Plex LS reagents on dried blood-spot assays Performs well with good assay analytical CV% on a Sciex API5000 instrument (also on an API3200) Capable of detecting true confirmed LS from an unaffected population In a limited cohort potential to distinguish carriers and pseudo deficiencies. Larger studies are required to confirm LC-MSMS method provides a confirmatory and diagnostic assay from dried blood-spots A solid phase solid-phase-extraction (SPE-silica) is also being investigated Proposal for a larger study to be conducted in the future Towards replacing existing 4-MU assays for diagnostic assays