Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots
Abstract
:1. Introduction
2. MS/MS and DMF-F Workflows
3. Screen Positive Rates
4. Assay Imprecision
5. Substrate Concentration and the Product Versus Time Progress Curve
6. Expansion of Assay Platforms to Additional LSDs
7. Concluding Remarks
Supplementary Materials
Acknowledgments
Conflicts of Interest
References
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Assay Step | Number of Liquid Transfers Per 96 Samples 1 | |
---|---|---|
DMF-F | Transfer enzyme extraction solvent to 96 well plate of DBS punches, seal plate, shake at room temperature for 30 min | 1 transfer with a 96-channel pipettor |
Load two microfluidics plates with filler fluid | 2 transfers | |
Load four calibrator solutions one by one to two microfluidics plates | 8 transfers | |
Load two wells to each of two microfluidics plates with stop buffer, one for each enzyme | 4 transfers | |
Load two wells to each of two microfluidics plates with enzyme assay cocktail, one for each enzyme | 4 transfers | |
Load samples to each of two microfluidics plates four at a time per column | 2 transfers with a 48 channel pipettor | |
Place microfluidics plates in plate reader | 0 | |
MS/MS | Remove single assay cocktail from storage and aliquot to 96-well plate containing DBS punches | 1 transfer with a 96-channel pipettor, 10 min |
Seal plate and place in thermostated incubator/shaker for 3 h to overnight | 0 transfers, | |
Add quench solvent to each well | 1 transfer with a 96-channel pipettor, 5 min | |
Transfer an aliquot of each well to a deep-well, 96-well plate | 1 transfer with a 96-channel pipettor, 5 min | |
Add ethyl acetate to each well | 1 transfer with a 96-channel pipettor, 1 min | |
Add water to each well | 1 transfer with a 96-channel pipettor, 1 min | |
Centrifuge for 5 min at room temperature | 0 transfers, 5 min | |
Transfer an aliquot of upper ethyl acetate layer to a shallow-well, 96-well plate | 1 transfer with a 96-channel pipettor, 5 min | |
Remove solvent with a multi-jet air dryer in fume hood | 0 transfers, 10 min | |
Add MS/MS infusion solvent to each well, seal plate and place on shaker at room temperature for 10 min | 1 transfer with a 96-channel pipettor, 6 min | |
Place 96-well plate in autosampler of MS/MS instrument | 0 transfers, 5 min |
NBS Laboratory | NBS Method | Current Cutoff | Number of Below Cutoff Samples | Number of below Cutoff Samples Per 100,000 Newborns | Source of Data |
---|---|---|---|---|---|
New York | MS/MS | 15% of mean GAA activity | 147 per 660,000 | 22 | C. Stevens, J. Orsini, New York DOH [15] |
Washington | MS/MS | 15% of mean GAA activity | 9 per 45,000 | 20 | [3,15] |
Ohio | MS/MS | 8.5% of mean GAA activity | 7 per 149,000 | 5 (32 if used 15% cutoff) | R. Hage, Ohio DOH [15] |
Illinois | MS/MS | 18% of mean GAA activity | 139 per 219,000 | 63 (14 if use 15% cutoff) | R. Shao, Illinois DOH; [9,15] |
Pennsylvania | MS/MS | 19% of mean GAA activity | 18 per 104,000 | 18 | Pennsylvannia DOH [15] |
Wisconsin | MS/MS | 15% of mean GAA activity | 1 per 8,250 | 12 | M. Baker, Wisconsin DOH [15] |
Tennessee | MS/MS | 15% of mean GAA activity | 2 per 12,279 | 16 | G. Dizikes, Tennessee DOH [15] |
Kentucky | MS/MS | Footnote 1 | 2 per 55,000 | 4 | [14] |
New York City | MS/MS | 15% of mean GAA activity | 6 per 18,105 | 33 | [16] |
Italy (Veneto Region) | MS/MS | 19% of mean GAA activity | 5 per 44,000 | 11 per 100,000 | [17] |
NBS Laboratory | NBS Method | Current Cutoff | Number of Below Cutoff Samples | Number of below Cutoff Samples Per 100,000 Newborns | Source of Data |
---|---|---|---|---|---|
New York | MS/MS | 10% of IDUA activity | 7 per 32,000 | 22 (20 if used 7.2%) 2 | (J. Orsini, New York DOH) [15] |
Washington | MS/MS | 10% of IDUA activity | 6 per 45,000 | 13 (7 if used 7.2%) | [3,15] |
Ohio | MS/MS | 7.2% of IDUA activity | 19 per 149,000 | 13 | (R. Hage, Ohio DOH) [15] |
Illinois | MS/MS | 28% of IDUA activity | 151 per 219,000 | 69 (11 if used 7.2%) | (R. Shao, Illinois DOH) [9,15] |
Kentucky | MS/MS | Footnote 1 | 1 per 55,000 | 2 | [14] |
New York City | MS/MS | 15% of mean IDUA activity | 13 per 35,816 | 36 2 | [16] |
Chinese Foundation of Health (Taiwan) | MS/MS | 7.2% of IDUA activity | 5 per 93,000 | 6 | (H-C. Liao, Chinese Foundation of Health) [15] |
National Taiwan University Hospital (Taiwan) | MS/MS | 15% of IDUA activity | 2 per 62,562 | 3 (3 if use 7.2%) | (P. Hwu, National Taiwan University Hospital) [15] |
Italy (Veneto region) | MS/MS | 19% IDUA activity | 8 per 44,000 | 18 per 100,000 | [17] |
LSD | MS/MS Assay | DMF-F Assay |
---|---|---|
Ceroid lipofuscinoisis I | [24] 2 | |
Ceroid lipofuscinoisis II | [4] 1 | |
Fabry | [3] | [2] |
Gaucher | [3] | [2] |
Krabbe | [3] | |
Metachromatic Leukodystrophy | [30] 2,3 | |
MPS-I | [3] | [2] |
MPS-II | [4] | [33] |
MPS-IIIA | [13] 2 | |
MPS-IIIB | [4] | |
MPS-IVA | [4] 2 | |
MPS-VI | [4] 2 | |
MPS-VII | [4] | |
Niemann-Pick-A/B | [3] | |
Niemann-Pick-C | [31] 2,4 | |
Pompe | [3] | [2] |
Wolman Disease | [23] |
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Gelb, M.H.; Lukacs, Z.; Ranieri, E.; Schielen, P.C.J.I. Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots. Int. J. Neonatal Screen. 2019, 5, 1. https://doi.org/10.3390/ijns5010001
Gelb MH, Lukacs Z, Ranieri E, Schielen PCJI. Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots. International Journal of Neonatal Screening. 2019; 5(1):1. https://doi.org/10.3390/ijns5010001
Chicago/Turabian StyleGelb, Michael H., Zoltan Lukacs, Enzo Ranieri, and Peter C. J. I. Schielen. 2019. "Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots" International Journal of Neonatal Screening 5, no. 1: 1. https://doi.org/10.3390/ijns5010001