Presentation on theme: "Screening for Lysosomal Storage Disorders Joseph Orsini, PhD"— Presentation transcript:
1 Screening for Lysosomal Storage Disorders Joseph Orsini, PhD Krabbe screeningPsychosine in DBS 3. Overview of LSD screening methods
2 LSD Assay Compounds Gelb et al, 2008 5-enzymes 3 Gaucher 1 Niemann-Pick2 Krabbe4 Fabry5 PompeGelb et al, 2008
3 New York State Krabbe Assay Punch 3-mm specimenAdd assay solution reagent and incubate19 hoursUp to 2496-wellplatesare testedeach dayQuench reaction (50/50 MeOH/EtAc)Liquid / liquid extraction (EtAc/H20)Remove organic phase (150 μL)Dry plates1 hourRe-dissolve in MS suitable solvent (80/20 MeOH/H20)Analyze samples, 1.5 minutes per sampleCalculate activity/sample, daily mean activity, % of daily mean act/sample
4 Age dependency data<24 hrSamples 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 meanRetested in duplicate (or more)Average of 3 samples ≤ 12%Average of 3 samples > 12%DNA testing1 or more mutationsNo mutationsScreen PositiveReferralScreen negative
9 New York State Newborn Screening for Krabbe Disease August 7, 2006 to May 31, 2012 DriedBloodSpot1,556,172 Screened for low GALC ActivityThreshold activity ≤12% daily meanHigh Throughput MS/MSDriedBloodSpotDNA sequencing startedsequencing completed191 low activity polymorphismsVenousBlood &Follow up261→ Counseling andVenous Confirmation25 mod. risk3H labeled - natural substrateηmol/h/mg proteinηmol/h/mg protein12 high risk(4 KD*)8 “healthy” up to 60 mos3 → HCT, 1 dead, 1 chronic hemolytic anemia1 → refused HSCT< 0.15 ηmol/h/mg protein*4KD Predicted to have infantile formBased on genotype and neurodiagnostic testing
10 Challenges/Lessons Learned 1. Number of newborn patients very limited-- Fresh, age-specific (newborn) controls necessary2. DOB samples have higher activities-- Ask for repeat3. Normalize data to daily run (high volume helps)-- Alleviate seasonal changes4. 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 GALCGalactosylcerebrosideGalactosylshingosine (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 GlucosylsphingosineUnknown peakDimethylsphingosineInternal 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 activityNewborns grouped as low-risk.Newborns with normal GALC activity in newborn screening.
14 Some observations1. 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 DiseaseIL/MO/NJ/NM: Mandated testing for Niemann-Pick, Krabbe, Gaucher, Fabry, Pompe (MPS1?).Illinois is pursuing HPLC-MS/MS methodMissouri is working with Advanced Liquid Logic methodWashington: 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.”
17 Potential solutions to method challenges: Eliminate first elution and distribution stepsDecrease 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 Incubate19 hrsAdd 100 µL1:1EtOAc/MeOHQuadruplex Solution30 µLAdd400 µL EtOAc& 400 µL H2OPlate 1APlate 1ADispense150 µLOrganic LayerPlate 1ACentrifugeRPMASM Solution30 µLAdd 100 µL1:1EtOAc/MeOHCombineLiq/LiqLiq/LiqPlate 1BPlate 1BPlate 1BDry and reconstitute w/ 150 µL 19:1 EtOAc/MeOHTransfer to flat-bottomSPEWash with200 µL19:1EtOAc/MeOHDry andReconstitute w/ 130 µL80:20MeOH/H2O w/ 5 mmol/L ammonium formateAnalyze withMS/MSSPEVery 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 stepsDecrease enzyme reactions from 5 to 2Simplified SPE step by decreasing amount of solvent needed – can be done unattended by a liquid handlerExtracts are very clean, so minimizes maintenance required on MS/MSNo need to perform chromatographyCan 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
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).
27 Advantages/Disadvantages 1. Simple sample prep. –- No sample extraction/distributionMinimal number of enzyme reactionsNo L/LNo SPEMay be possible to add substrates to analysis (psychosine, glucosyl sphingosine, others?)ExpandableDisadvantage: Uses HPLC which may be more complex. However, HPLC is used in labs routinely for hemoglobinopathy screening.
28 Fluorometric assaysSingle 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 platformDisadvantages:- 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 UPLCMS/MS: with UPLCChoose between a more complex preparation/clean-up or more complex instrumental set-upConsiderations:Depends on what disorders you want to test now and what may be added in the futurePersonnel capabilitiesStart-up costs