New York State Krabbe Assay Punch 3-mm specimen Add assay solution reagent and incubate Quench reaction (50/50 MeOH/EtAc) Liquid / liquid extraction (EtAc/H20) Remove organic phase (150 μ L ) Dry plates Re-dissolve in MS suitable solvent (80/20 MeOH/H20) 19 hours Analyze samples, 1.5 minutes per sample 1 hour Up to well plates are tested each day Calculate activity/sample, daily mean activity, % of daily mean act/sample
Age dependency data Samples taken <24 hours considered invalid, repeat specimen is requested. <24 hr
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.
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% Screen negative Screen Positive Referral DNA testing 1 or more mutationsNo mutations
Krabbe <20 Retest and <12% - Monthly Rates
Krabbe Referral/Polymorphism – Monthly Rates
New York State Newborn Screening for Krabbe Disease August 7, 2006 to May 31, ,556,172 Screened for low GALC Activity Threshold activity ≤12% daily mean High Throughput MS/MS 601 DNA sequencing started 452 sequencing completed 191 low activity polymorphisms 261→ Counseling and Venous Confirmation 25 mod. risk 12 high risk (4 KD*) 3 H labeled - natural substrate η mol/h/mg protein ηmol/h/mg protein Dried Blood Spot Dried Blood Spot Venous Blood & Follow up 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
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
Krabbe disease: Psychosine as a marker? 1. Psychosine is another substrate of GALC a.Galactosylcerebroside b.Galactosylshingosine (Psychosine) 2.Psychosine is thought to be the substrate most responsible for disease progression. 3.Interestingly, in Gaucher the neuronopathic form of Gaucher disease is thought to be caused by accumulation of glucosylsphingosine. 4.In Fabry: “Elevated globotriaosylsphingosine is hallmark of Fabry disease.”
Psychosine Data Plot Legend A.Krabbe control specimens, varied age of onset, mostly late onset patients (from older patients). B.Newborns identified with infantile Krabbe disease via newborn screening. C.Newborns grouped as “high risk” due to very low GALC activity and mutation analyses that are asymptomatic, and being followed. D.Newborns grouped as “moderate risk,” based on GALC activity E.Newborns grouped as low-risk. F.Newborns with normal GALC activity in newborn screening.
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.
Overview of LSD’s Testing in U.S. 1.New York State: Krabbe Disease 2.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 3.Washington: Pilot study for Fabry, MPS-1, and Pompe disease (the “treatable three”). Is using Advancing UPLC-MS/MS method. 4. Secretary Advisory Board on Heritable Diseases in Newborns and Children (SACHDNC) has recommended that Pompe and MPS-1 undergo further evidence-based review. 5.Moving Target. Adding test to MS/MS panel not as easy as “turning on a new channel.”
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).
NYS modified 5-plex method Incubate 19 hrs Dispense 150 µL Organic Layer Add 400 µL EtOAc & 400 µL H 2 O Add 100 µL 1:1 EtOAc/MeOH Plate 1A Quadruplex Solution 30 µL Liq/Liq SPE Wash with 200 µL 19:1 EtOAc/MeOH SPE Centrifuge RPM Dry and reconstitute w/ 150 µL 19:1 EtOAc/MeOH Dry and Reconstitute w/ 130 µL 80:20 MeOH/H 2 O w/ 5 mmol /L ammonium formate Plate 1B ASM Solution 30 µL Add 100 µL 1:1 EtOAc/MeOH Combine Analyze with MS/MS Transfer to flat-bottom Plate 1A Plate 1B Plate 1A Plate 1B 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.
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
Population Results 4 plus 1 Multiplex
Population Study: Highlights 1. Completed this study in one week (5,055 specimens - one week of specimens in our lab). 2.One analyst performed all of the work (in addition to normal duties). 3.All specimens were tested on one MS/MS (we would expect to use three routinely) 4.The solid phase extraction was automated and we only use 200 μ L of solvent, not 1600 μ L. 5.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).
LSD Screening: HPLC/MS/MS Methods
LSD Screening: Original Five Substrates
LSD Screening: Four more (9-Plex)
Advantages/Disadvantages Advantages: 1. Simple sample prep. – - No sample extraction/distribution - Minimal number of enzyme reactions - No L/L - No SPE 2. May be possible to add substrates to analysis (psychosine, glucosyl sphingosine, others?) 3. Expandable Disadvantage: Uses HPLC which may be more complex. However, HPLC is used in labs routinely for hemoglobinopathy screening.
Fluorometric assays 1.Single enzyme reactions: available for Pompe, Fabry, MPS-1, Gaucher (not Krabbe or NP-AB). Can only test one enzyme at a time. 2.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
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 3.Choose between a more complex preparation/clean-up or more complex instrumental set-up Considerations: 1.Depends on what disorders you want to test now and what may be added in the future 2.Personnel capabilities 3.Start-up costs
Thank you - questions?
Other Options are available Liquidator 96 (Rainin): University of Washington uses this.