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

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.

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Digital Microfluidics in Newborn Screening for Mucopolysaccharidoses: A Progress Report

International Journal of Neonatal Screening ◽

10.3390/ijns6040078 ◽

2020 ◽

Vol 6 (4) ◽

pp. 78

Author(s):

Jon Washburn ◽

David S. Millington

Keyword(s):

Newborn Screening ◽

Digital Microfluidics ◽

Residual Activity ◽

Practical Experience ◽

The United States ◽

Dried Blood Spots ◽

Type I ◽

The Usa ◽

Digital Microfluidic ◽

Mps I

Newborn screening (NBS) for mucopolysaccharidosis type I (MPS I, Hurler syndrome) is currently conducted in about two-fifths of the NBS programs in the United States and in a few other countries. Screening is performed by measurement of residual activity of the enzyme alpha-l-iduronidase in dried blood spots using either tandem mass spectrometry or digital microfluidic fluorometry (DMF). In this article, we focus on the development and practical experience of using DMF to screen for MPS I in the USA. By means of their responses to a questionnaire, we determined for each responding program that is screening for MPS I using DMF the screen positive rate, follow-up methods, and classification of confirmed cases as either severe or attenuated. Overall, the results show that at the time of reporting, over 1.3 million newborns in the US were screened for MPS I using DMF, 2094 (0.173%) of whom were screen positive. Of these, severe MPS I was confirmed in five cases, attenuated MPS I was confirmed in two cases, and undetermined phenotype was reported in one case. We conclude that DMF is an effective and economical method to screen for MPS I and recommend second-tier testing owing to high screen positive rates. Preliminary results of NBS for MPS II and MPS III using DMF are discussed.

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

International Journal of Neonatal Screening ◽

10.3390/ijns5010001 ◽

2018 ◽

Vol 5 (1) ◽

pp. 1 ◽

Cited By ~ 15

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.

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Digital microfluidics using a differentially polarized interface (DPI) to enhance translational force

Lab on a Chip ◽

10.1039/c8lc00652k ◽

2018 ◽

Vol 18 (21) ◽

pp. 3293-3302 ◽

Cited By ~ 3

Author(s):

Md Enayet Razu ◽

Jungkyu Kim

Keyword(s):

Low Voltage ◽

Digital Microfluidics ◽

Microfluidic Platform ◽

Digital Microfluidic

A low-voltage and differentially polarized digital microfluidic platform is developed by enhancing the electromechanical force for droplet translation.

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Treatment Efficiency in Gaucher Patients Can Reliably Be Monitored by Quantification of Lyso-Gb1 Concentrations in Dried Blood Spots

International Journal of Molecular Sciences ◽

10.3390/ijms21134577 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4577 ◽

Cited By ~ 4

Author(s):

Claudia Cozma ◽

Paskal Cullufi ◽

Guido Kramp ◽

Marina Hovakimyan ◽

Virtut Velmishi ◽

...

Keyword(s):

Early Stage ◽

Subsequent Development ◽

High Sensitivity ◽

Dried Blood Spots ◽

Lysosomal Storage ◽

Treatment Efficiency ◽

Blood Spots ◽

Enzyme Replacement ◽

Treatment Conditions ◽

Clinical Consequences

Gaucher disease (GD) is a lysosomal storage disorder that responds well to enzyme replacement therapy (ERT). Certain laboratory parameters, including blood concentration of glucosylsphingosine (Lyso-Gb1), the lyso-derivate of the common glycolipid glucocerebroside, correlate with clinical improvement and are therefore considered candidate-monitoring biomarkers. Whether they can indicate a reduction or loss of treatment efficiency, however, has not been systematically addressed for obvious reasons. We established and validated measurement of Lyso-Gb1 from dried blood spots (DBSs) by mass spectrometry. We then characterized the assay’s longitudinal performance in 19 stably ERT-treated GD patients by dense monitoring over a 3-year period. The observed level of fluctuation was accounted for in the subsequent development of a unifying data normalization concept. The resulting approach was eventually applied to data from Lyso-Gb1 measurements after an involuntary treatment break for all 19 patients. It enabled separation of the “under treatment” versus “not under treatment” conditions with high sensitivity and specificity. We conclude that Lyso-Gb1 determination from DBSs indicates treatment issues already at an early stage before clinical consequences arise. In addition to its previously shown diagnostic utility, Lyso-Gb1 thereby qualifies as a monitoring biomarker in GD patients.

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Neonatal Screening for MPS Disorders in Latin America: A Survey of Pilot Initiatives

International Journal of Neonatal Screening ◽

10.3390/ijns6040090 ◽

2020 ◽

Vol 6 (4) ◽

pp. 90

Author(s):

Francyne Kubaski ◽

Inês Sousa ◽

Tatiana Amorim ◽

Danilo Pereira ◽

Joe Trometer ◽

...

Keyword(s):

Latin America ◽

Newborn Screening ◽

Treatment Options ◽

Digital Microfluidics ◽

Premature Death ◽

Lysosomal Storage ◽

Pilot Studies ◽

The Usa ◽

History Of ◽

Mps I

Newborn screening enables the diagnosis of treatable disorders at the early stages, and because of its countless benefits, conditions have been continuously added to screening panels, allowing early intervention, aiming for the prevention of irreversible manifestations and even premature death. Mucopolysaccharidoses (MPS) are lysosomal storage disorders than can benefit from an early diagnosis, and thus are being recommended for newborn screening. They are multisystemic progressive disorders, with treatment options already available for several MPS types. MPS I was the first MPS disorder enrolled in the newborn screening (NBS) panel in the USA and a few other countries, and other MPS types are expected to be added. Very few studies about NBS for MPS in Latin America have been published so far. In this review, we report the results of pilot studies performed in Mexico and Brazil using different methodologies: tandem mass spectrometry, molecular analysis, digital microfluidics, and fluorimetry. These experiences are important to report and discuss, as we expect to have several MPS types added to NBS panels shortly. This addition will enable timely diagnosis of MPS, avoiding the long diagnostic odyssey that is part of the current natural history of this group of diseases, and leading to a better outcome for the affected patients.

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Cardiac Murmur in a Boy with Normal Paternal Prenatal Carrier Screening for Pompe Disease

Case Reports in Pediatrics ◽

10.1155/2019/6274979 ◽

2019 ◽

Vol 2019 ◽

pp. 1-3

Author(s):

Allison M. Jay ◽

Premchand Anne ◽

David Stockton

Keyword(s):

Newborn Screening ◽

Pompe Disease ◽

Autosomal Recessive ◽

Genetic Test ◽

Carrier Screening ◽

Lysosomal Storage ◽

Cardiac Murmur ◽

Enzyme Replacement ◽

Prenatal Test ◽

Infantile Type

Introduction. Pompe disease is an autosomal recessive lysosomal storage disorder with marked morbidity and mortality, if untreated. With the advent of enzyme replacement therapy, it is essential to identify the infantile-type as early as possible to mitigate the effects of the enzyme deficiency. Identification is possible prenatally with testing of both parents. More recently, many states have instituted newborn screening for this condition. Case. We report a patient with infantile-onset Pompe disease with a normal paternal carrier genetic test, born prior to newborn screening for Pompe disease in the state of Michigan. The infant’s father was retested once the infant was diagnosed with Pompe disease postnatally and noted to have a mutation conducive to Pompe disease. Conclusion. Providers should have a strong clinical suspicion for disorders even if prenatal parental carrier screening is normal. A normal parental prenatal test does not exclude the possibility that the fetus may be diagnosed with a disorder postnatally, and pediatricians may be faced with limitations in accuracy of parents’ recollection of parental testing results.

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Digital microfluidics for spheroid-based invasion assays

Lab on a Chip ◽

10.1039/c5lc01569c ◽

2016 ◽

Vol 16 (8) ◽

pp. 1505-1513 ◽

Cited By ~ 21

Author(s):

Brian F. Bender ◽

Andrew. P. Aijian ◽

Robin. L. Garrell

Keyword(s):

Drug Discovery ◽

Cell Invasion ◽

Cell Biology ◽

Digital Microfluidics ◽

Three Dimensional ◽

Multicellular Spheroids ◽

Microfluidic Platform ◽

Digital Microfluidic ◽

Biology Research ◽

Invasion Assays

A digital microfluidic platform that enables the formation, gel encapsulation, and assaying of three-dimensional multicellular spheroids is described. Such a platform can facilitate automation of cell invasion assays for cell biology research and drug discovery.

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Cholesteryl ester storage disease: a rare and possibly treatable cause of premature vascular disease and cirrhosis

Journal of Clinical Pathology ◽

10.1136/jclinpath-2012-201302 ◽

2013 ◽

Vol 66 (11) ◽

pp. 918-923 ◽

Cited By ~ 32

Author(s):

Tim Reynolds

Keyword(s):

Enzyme Activity ◽

Cholesteryl Ester ◽

Storage Disease ◽

Treatment Options ◽

Failure To Thrive ◽

Early Death ◽

Dried Blood Spots ◽

Lysosomal Storage ◽

Enzyme Replacement ◽

Reduced Activity

Cholesteryl ester storage disease (CESD) is an autosomal recessive lysosomal storage disorder caused by a variety of mutations of the LIPA gene. These cause reduced activity of lysosomal acid lipase, which results in accumulation of cholesteryl esters in lysosomes. If enzyme activity is very low/absent, presentation is in infancy with failure to thrive, malabsorption, hepatosplenomegaly and rapid early death (Wolman disease). With higher but still low enzyme activity, presentation is later in life with hepatic fibrosis, dyslipidaemia and early atherosclerosis.Identification of this rare disorder is difficult as it is essential to assay leucocyte acid phosphatase activity. An assay using specific inhibitors has now been developed that facilitates measurement in dried blood spots. Treatment of CESD has until now been limited to management of the dyslipidaemia, but this does not influence the liver effects. A new enzyme replacement therapy (Sebelipase) has now been developed that could change treatment options for the future.

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