scholarly journals Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots

2018 ◽  
Vol 5 (1) ◽  
pp. 1 ◽  
Author(s):  
Michael Gelb ◽  
Zoltan Lukacs ◽  
Enzo Ranieri ◽  
Peter Schielen
Keyword(s):  
Newborn Screening ◽  
Digital Microfluidics ◽  
Lysosomal Storage Diseases ◽  
Dried Blood Spots ◽  
Enzymatic Activities ◽  
Lysosomal Storage ◽  
Blood Spots ◽  
Positive Rate ◽  
Control And Prevention ◽  
Case Basis

All worldwide newborn screening (NBS) for lysosomal storage diseases (LSDs) is performed as a first-tier test by measurement of lysosomal enzymatic activities in dried blood spots (DBS). The currently two available methodologies used for measurement of enzymatic activities are tandem mass spectrometry (MS/MS) and digital microfluidics fluorimetry (DMF-F). In this chapter we summarize the workflows for the two platforms. Neither platform is fully automated, but the relative ease of workflow will be dependent upon the specific operation of each newborn screening laboratory on a case-by-case basis. We provide the screen positive rate (the number of below cutoff newborns per 100,000 newborns) from all NBS laboratories worldwide carrying out MS/MS-based NBS of one or more LSDs. The analytical precision of the MS/MS method is higher than that for DMF-F as shown by analysis of a common set of quality control DBS by the Centers for Disease Control and Prevention (CDC). Both the MS/MS and DMF-F platforms enable multiplexing of the LSD enzymes. An advantage of MS/MS over DMF-F is the ability to include assays of enzymatic activities and biomarkers for which no fluorimetric methods exist. Advantages of DMF-F over MS/MS are: (1) simple to use technology with same-day turn-around time for the lysosomal enzymes with the fastest rates compared to MS/MS requiring overnight analytical runs.; (2) the DMF-F instrumentation, because of its simplicity, requires less maintenance than the MS/MS platform.

scholarly journals Digital Microfluidic Platform for Multiplexing Enzyme Assays: Implications for Lysosomal Storage Disease Screening in Newborns

2011 ◽  
Vol 57 (10) ◽  
pp. 1444-1451 ◽  
Author(s):  
Ramakrishna S Sista ◽  
Allen E Eckhardt ◽  
Tong Wang ◽  
Carrie Graham ◽  
Jeremy L Rouse ◽  
...  
Keyword(s):  
Newborn Screening ◽  
Incubation Time ◽  
Digital Microfluidics ◽  
Dried Blood Spots ◽  
Reference Laboratory ◽  
Enzymatic Analysis ◽  
Lysosomal Storage ◽  
Microfluidic Platform ◽  
Enzyme Replacement ◽  
Digital Microfluidic

BACKGROUND Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. METHODS We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. RESULTS Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. CONCLUSIONS A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

scholarly journals Hurler-like Phenotype

2001 ◽  
Vol 47 (12) ◽  
pp. 2098-2102 ◽  
Author(s):  
Néstor A Chamoles ◽  
Mariana B Blanco ◽  
Daniela Gaggioli ◽  
Carina Casentini
Keyword(s):  
Enzyme Activities ◽  
Lysosomal Storage Diseases ◽  
Dried Blood Spots ◽  
Lysosomal Storage ◽  
Testing Laboratory ◽  
Storage Diseases ◽  
Blood Spots ◽  
Sample Stability ◽  
Mucolipidosis Ii ◽  
Radioactive Substrate

Abstract Background: Clinical differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype. Methods: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 °C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers. Results: Intra- and interassay CVs were <9% and <15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 °C were ≤27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls. Conclusions: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.

Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots

2011 ◽  
Vol 102 (1) ◽  
pp. 44-48 ◽  
Author(s):  
L.F. Oemardien ◽  
A.M. Boer ◽  
G.J.G. Ruijter ◽  
A.T. van der Ploeg ◽  
J.B.C. de Klerk ◽  
...  
Keyword(s):  
Lysosomal Storage Diseases ◽  
Dried Blood Spots ◽  
Lysosomal Storage ◽  
Storage Diseases ◽  
Diagnostic Assays ◽  
Blood Spots

scholarly journals The Importance of Assay Imprecision near the Screen Cutoff for Newborn Screening of Lysosomal Storage Diseases

2019 ◽  
Vol 5 (2) ◽  
pp. 17 ◽  
Author(s):  
Bruce Robinson ◽  
Michael Gelb
Keyword(s):  
Newborn Screening ◽  
Lysosomal Storage Diseases ◽  
Dried Blood Spots ◽  
False Positives ◽  
Theoretical Treatment ◽  
Lysosomal Storage ◽  
Cutoff Value ◽  
Storage Diseases ◽  
Screen Performance ◽  
False Positives And Negatives

For newborn screening (NBS) of lysosomal storage diseases, programs measure enzymatic activities in dried blood spots (DBS) and, in most cases, act on samples where the measurement is below a specific cutoff value. The rate of false positives and negatives in any NBS program is of critical importance. The measured values across a population of newborns are governed by many factors, and in this article we focus on assay imprecision. Assay parameters including the Analytical Range and the Z-Factor have been discussed as a way to compare assay performance for NBS of lysosomal storage diseases. Here we show that these parameters are not rigorously connected to the rate of false positives and negatives. Rather, it is the assay imprecision near the screen cutoff that is the most important parameter that determines the rate of false positives and negatives. We develop the theoretical treatment of assay imprecision and how it is linked to screen performance. What emerges is a useful type of parametric plot that allows for rigorous assessment of the effect of assay imprecision on the rate of false positives and false negatives that is independent of the choice of screen cutoff value. Such plots are useful in choosing cutoff values. They also show that a high assay imprecision cannot be overcome by changing the cutoff value or by use of postanalysis, statistical tools. Given the importance of assay imprecision near the cutoff, we propose that quality control DBS are most useful if they span a range of analyte values near the cutoff. Our treatment is also appropriate for comparing the performance of multiple assay platforms that each measure the same quantity (i.e., the enzymatic activity in DBS). The analysis shows that it is always best to use the assay platform that gives the lowest imprecision near the cutoff.

Sign in / Sign up

Export Citation Format

Share Document