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Screening for Lysosomal Storage Disorders Joseph Orsini, PhD

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Presentation on theme: "Screening for Lysosomal Storage Disorders Joseph Orsini, PhD"— Presentation transcript:

1 Screening for Lysosomal Storage Disorders Joseph Orsini, PhD
Krabbe screening Psychosine in DBS 3. Overview of LSD screening methods

2 LSD Assay Compounds Gelb et al, 2008 5-enzymes 3 Gaucher
1 Niemann-Pick 2 Krabbe 4 Fabry 5 Pompe Gelb et al, 2008

3 New York State Krabbe Assay
Punch 3-mm specimen Add assay solution reagent and incubate 19 hours Up to 24 96-well plates are tested each day Quench reaction (50/50 MeOH/EtAc) Liquid / liquid extraction (EtAc/H20) Remove organic phase (150 μL) Dry plates 1 hour Re-dissolve in MS suitable solvent (80/20 MeOH/H20) Analyze samples, 1.5 minutes per sample Calculate activity/sample, daily mean activity, % of daily mean act/sample

4 Age dependency data <24 hr Samples taken <24 hours considered invalid, repeat specimen is requested.

5 Algorithm considerations
Looking for low activity samples when majority of samples show high activity (cross-contamination issues). Need accuracy at low end of calibration (blank subtraction. Apparent overlap in carriers and diseased population (DNA testing). Average activity of Krabbe controls is 5% of daily mean (these were older patients). Consider specimens collected on day of birth unsuitable unless below cutoff. Fast turnaround is required.

6 Krabbe Screening: Cutoffs and Testing Algorithm
All specimens tested for GALC activity < 20% of daily mean > 20% of daily mean Retested in duplicate (or more) Average of 3 samples ≤ 12% Average of 3 samples > 12% DNA testing 1 or more mutations No mutations Screen Positive Referral Screen negative

7 Krabbe <20 Retest and <12% - Monthly Rates

8 Krabbe Referral/Polymorphism – Monthly Rates

9 New York State Newborn Screening for Krabbe Disease August 7, 2006 to May 31, 2012
Dried Blood Spot 1,556,172 Screened for low GALC Activity Threshold activity ≤12% daily mean High Throughput MS/MS Dried Blood Spot DNA sequencing started sequencing completed 191 low activity polymorphisms Venous Blood & Follow up 261→ Counseling and Venous Confirmation 25 mod. risk 3H labeled - natural substrate ηmol/h/mg protein ηmol/h/mg protein 12 high risk (4 KD*) 8 “healthy” up to 60 mos 3 → HCT, 1 dead, 1 chronic hemolytic anemia 1 → refused HSCT < 0.15 ηmol/h/mg protein *4KD Predicted to have infantile form Based on genotype and neurodiagnostic testing

10 Challenges/Lessons Learned
1. Number of newborn patients very limited -- Fresh, age-specific (newborn) controls necessary 2. DOB samples have higher activities -- Ask for repeat 3. Normalize data to daily run (high volume helps) -- Alleviate seasonal changes 4. Overlap of enzyme activity (mutations v. polymorphisms) -- DNA analysis reduces referrals by >40% 5. Genotype does not always predict phenotype

11 Krabbe disease: Psychosine as a marker?
Psychosine is another substrate of GALC Galactosylcerebroside Galactosylshingosine (Psychosine) Psychosine is thought to be the substrate most responsible for disease progression. Interestingly, in Gaucher the neuronopathic form of Gaucher disease is thought to be caused by accumulation of glucosylsphingosine. In Fabry: “Elevated globotriaosylsphingosine is hallmark of Fabry disease.”

12 Psychosine Testing Method: HPLC-MS/MS
Glucosylsphingosine Unknown peak Dimethylsphingosine Internal standard

13 Psychosine Data Plot Legend
Krabbe control specimens, varied age of onset, mostly late onset patients (from older patients). Newborns identified with infantile Krabbe disease via newborn screening. Newborns grouped as “high risk” due to very low GALC activity and mutation analyses that are asymptomatic, and being followed . Newborns grouped as “moderate risk,” based on GALC activity Newborns grouped as low-risk. Newborns with normal GALC activity in newborn screening.

14 Some observations 1. Psychosine can be detected in dried blood spots using highly sensitive HPLC MS/MS method. 2. Psychosine is very elevated in newborns who were predicted to have infantile Krabbe disease. 3. The psychosine concentrations in non-symptomatic high and moderate risk categories are only slightly higher than those in the low-risk and no-risk newborns.

15 Overview of LSD’s Testing in U.S.
New York State: Krabbe Disease IL/MO/NJ/NM: Mandated testing for Niemann-Pick, Krabbe, Gaucher, Fabry, Pompe (MPS1?). Illinois is pursuing HPLC-MS/MS method Missouri is working with Advanced Liquid Logic method Washington: Pilot study for Fabry, MPS-1, and Pompe disease (the “treatable three”). Is using Advancing UPLC-MS/MS method. Secretary Advisory Board on Heritable Diseases in Newborns and Children (SACHDNC) has recommended that Pompe and MPS-1 undergo further evidence-based review. Moving Target. Adding test to MS/MS panel not as easy as “turning on a new channel.”

16 Previous MS/MS Screening Method
Initial extraction/distributions steps Reduce number of reactions Incubate 19 hrs Add 100 µL 1:1 EtOAc/MeOH Dispense 10 µL GAA 15 µL GAA Dispense 100 µL Add 70 µL Extraction Buffer Multiplex Extraction 1 hr Add 400 µL EtOAc & 400 µL H2O GLA 15 µL GLA Centrifuge RPM Plate 1A Plate 1A ABG Liq/Liq ABG 15 µL ASM 15 µL ASM GALC 30 µL GALC Plate 1B Plate 1B Plate 1B Eliminate Liq/Liq and SPE (HPLC) Dry and reconstitute w/ 100 µL 19:1 EtOAc/MeOH Dispense 300 µL Organic Layer Dispense 100 µL SPE Wash with 1600 µL 19:1 EtOAc/MeOH Dry and Reconstitute w/ 200 µL 80:20 MeOH/H2O w/ 5 mmol/L ammonium formate Analyze with MS/MS Liq/Liq Pre-SPE SPE

17 Potential solutions to method challenges:
Eliminate first elution and distribution steps Decrease the number of enzyme reactions and simplify the SPE step (New York State approach). Perform sample cleanup in-line using chromatograph (Italy/Austria approach). Combine both approaches – decrease enzyme reactions and use HPLC for in-line clean-up (Mike Gelb/U. Washington). Other methods: Fluorescent substrates (Advanced Liquid Logic).

18 NYS modified 5-plex method
Incubate 19 hrs Add 100 µL 1:1 EtOAc/MeOH Quadruplex Solution 30 µL Add 400 µL EtOAc & 400 µL H2O Plate 1A Plate 1A Dispense 150 µL Organic Layer Plate 1A Centrifuge RPM ASM Solution 30 µL Add 100 µL 1:1 EtOAc/MeOH Combine Liq/Liq Liq/Liq Plate 1B Plate 1B Plate 1B Dry and reconstitute w/ 150 µL 19:1 EtOAc/MeOH Transfer to flat-bottom SPE Wash with 200 µL 19:1 EtOAc/MeOH Dry and Reconstitute w/ 130 µL 80:20 MeOH/H2O w/ 5 mmol/L ammonium formate Analyze with MS/MS SPE Very important note: the SPE step not only eliminates buffer components but minimizes the amount of substrate in final solution that is analyzed on the MS/MS.

19 Method Advantages Advantages:
Eliminated elution and distributions steps Decrease enzyme reactions from 5 to 2 Simplified SPE step by decreasing amount of solvent needed – can be done unattended by a liquid handler Extracts are very clean, so minimizes maintenance required on MS/MS No need to perform chromatography Can add Hurlers (MPS1) Disadvantages: Still need to do L/L (simple) and SPE (complicated) clean-ups! Still requires many plates to be used in processing of specimens

20 Population Results 4 plus 1 Multiplex

21 Population Study: Highlights
1. Completed this study in one week (5,055 specimens - one week of specimens in our lab). One analyst performed all of the work (in addition to normal duties). All specimens were tested on one MS/MS (we would expect to use three routinely) The solid phase extraction was automated and we only use 200 μL of solvent, not 1600μL. Equivalent of about three weeks worth of specimens all in one week. The MS/MS was cleaner than what we see under a normal week of testing Krabbe specimens (which do not have solid-phase extraction).

22 LSD Screening: HPLC/MS/MS Methods

23 LSD Screening: Original Five Substrates

24 LSD Screening: Four more (9-Plex)

25 Nine-Plex


27 Advantages/Disadvantages
1. Simple sample prep. – - No sample extraction/distribution Minimal number of enzyme reactions No L/L No SPE May be possible to add substrates to analysis (psychosine, glucosyl sphingosine, others?) Expandable Disadvantage: Uses HPLC which may be more complex. However, HPLC is used in labs routinely for hemoglobinopathy screening.

28 Fluorometric assays Single enzyme reactions: available for Pompe, Fabry, MPS-1, Gaucher (not Krabbe or NP-AB). Can only test one enzyme at a time. Advanced Liquid Logic – Advantages: - Micro-scale analysis – uses only one 3 mm punch - Equipment is automatable and simple to use - No MS/MS - Cheaper start-up costs - Smaller platform Disadvantages: - Can not run all enzymes - Still unproven in NBS lab

29 Three Approaches/How to choose?
Choose between a more complex preparation/clean-up or more complex instrumental set-up. MS/MS versus fluorometric? MS/MS: no UPLC MS/MS: with UPLC Choose between a more complex preparation/clean-up or more complex instrumental set-up Considerations: Depends on what disorders you want to test now and what may be added in the future Personnel capabilities Start-up costs

30 Thank you - questions?


32 Other Options are available
Liquidator 96 (Rainin): University of Washington uses this.

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