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.