Elsevier

Clinica Chimica Acta

Volume 424, 23 September 2013, Pages 12-18
Clinica Chimica Acta

Multiplex newborn screening for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases using a digital microfluidic platform

https://doi.org/10.1016/j.cca.2013.05.001Get rights and content

Highlights

  • Five-plex assay performed on a disposable cartridge capable of handling up to 44 samples

  • Multiplex analysis validated for Pompe, Fabry, Hunter, Gaucher and Hurler

  • All 5 enzymatic assays are completed from a single dried blood spot punch.

  • Punch to results within 3 h for rapid and high throughput analysis

Abstract

Purpose

New therapies for lysosomal storage diseases (LSDs) have generated interest in screening newborns for these conditions. We present performance validation data on a digital microfluidic platform that performs multiplex enzymatic assays for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases.

Methods

We developed an investigational disposable digital microfluidic cartridge that uses a single dried blood spot (DBS) punch for performing a 5-plex fluorometric enzymatic assay on up to 44 DBS samples. Precision and linearity of the assays were determined by analyzing quality control DBS samples; clinical performance was determined by analyzing 600 presumed normal and known affected samples (12 for Pompe, 7 for Fabry and 10 each for Hunter, Gaucher and Hurler).

Results

Overall coefficient of variation (CV) values between cartridges, days, instruments, and operators ranged from 2 to 21%; linearity correlation coefficients were ≥ 0.98 for all assays. The multiplex enzymatic assay performed from a single DBS punch was able to discriminate presumed normal from known affected samples for 5 LSDs.

Conclusions

Digital microfluidic technology shows potential for rapid, high-throughput screening for 5 LSDs in a newborn screening laboratory environment. Sample preparation to enzymatic activity on each cartridge is less than 3 h.

Introduction

Newborn screening (NBS) is generally performed for those diseases for which both an inexpensive screening test and treatment are available and the disease, if untreated, leads to profound morbidity or mortality. Nationally, newborn screening began with a screen for phenylketonuria [1], and has since expanded to include > 50 conditions [2] due to available treatments and technological advances in screening, such us multiplex tandem mass spectrometry (MS/MS) [3]. Lysosomal storage diseases (LSDs) have been identified as diseases where patients can greatly benefit from newborn screening. Although individual LSDs are rare, as a group they can lead to devastating consequences, including infant mortality [4], and many can be screened using dried blood spots (DBSs) [5], [6]. The combined incidence rate of LSDs is reported to be as high as 1:2315 live births [7]. Recent advances in treatment, including enzyme replacement therapy [8], for certain lysosomal storage diseases have generated renewed interest in newborn screening for individual LSDs [9], [10]. Here, we focus on a multiplex method using a single DBS punch to screen for five LSDs with proven treatments: Pompe disease (glycogen storage disease type II, caused by acid α-glucosidase (GAA) deficiency), Fabry disease (α-galactosidase (GLA) deficiency), Hunter disease (mucopolysaccharidosis type II, iduronate-2-sulfatase (IDS) deficiency), Gaucher disease (glucocerebrosidase (GBA) deficiency), and Hurler disease (mucopolysaccharidosis type I, α-iduronidase (IDU) deficiency).

At present, only 5 states in the USA (including New York, Illinois, Missouri, New Mexico, and New Jersey) have mandated newborn screening for select LSDs. Currently, the technologies available to perform DBS assays for lysosomal storage diseases are tandem mass spectrometry [11], microplate fluorometry [12], immunoassays [13] and, more recently, digital microfluidic fluorometry [14]. As more states move to adopt LSD screening, there is a need to develop rapid, efficient and economical high-throughput methods to screen for multiple LSDs simultaneously [15], [16], [17]. Orsini et al. reported using a 4 + 1 multiplex tandem MS/MS platform to screen for Gaucher, Pompe, Krabbe, Fabry, and Niemann-Pick A/B that reduced processing time and test complexity [18]. Mechtler et al. reduced tandem MS/MS incubation times to less than 4 h using a multiplex assay for Pompe, Fabry, Hunter, Gaucher, and Niemann-Pick A/B [15].

We have previously demonstrated that digital microfluidics can be used to screen for Hunter, Pompe and Fabry diseases using low throughput disposable cartridges [14], [19], [20]. A prototype cartridge designed by Advanced Liquid Logic, Inc. (ALL) that performed 3 assays (Pompe, Fabry and Gaucher) on up to 12 samples was recently pilot tested in Illinois Department of Public Health laboratories [21]. From a total of 8012 DBS samples screened, seven cases of Fabry and two cases of Gaucher disease were confirmed.

In this report, we demonstrate the capabilities of a new digital microfluidic system to meet the high throughput and rapid turnaround requirements of a newborn screening laboratory by means of a 5-plex fluorometric enzymatic assay for Pompe, Fabry, Hunter, Gaucher and Hurler diseases on a cartridge that accepts 44 specimens. The platform utilizes a single disposable cartridge that automates all liquid handling steps and reduces the overall time to result to < 3 h, using a single 3 mm DBS punch from each of the 44 specimens.

Section snippets

Dried blood spot samples

We obtained presumed normal, de-identified dried blot spots (NBS cards) from the North Carolina Division of Public Health NBS laboratories under a material transfer agreement. These spots were 2–3 months old and were stored at − 20 °C upon receipt. Duke University Biochemical Genetics Laboratory (Durham, NC), Shire Human Genetic Therapies, Inc. (Lexington, MA) and the Centers for Disease Control and Prevention (Atlanta, GA; CDC) laboratories provided de-identified affected DBS for Pompe (n = 12),

Precision

To determine day-to-day precision, mean enzymatic activities for each QC level for each instrument and each day were calculated. Overall mean, standard deviation, and % CV were calculated for each level. Enzymatic activity values presented in Table 1 are an average of the data points obtained over the eight instruments and three days. The instrument to instrument variation ranged between 6% and 21.3% for QCH, 5.9% and 23.6% for QCM and 13.2% and 25.2% for QCL for all 5 assays. Similarly the day

Conclusions

The results presented here represent important steps towards the validation of a multiplex, automated digital microfluidic platform to screen for five lysosomal storage diseases on a single disposable cartridge. The assay uses a single 3 mm punch from each of 44 DBS samples to generate results within 3 h on a single analyzer. In order to achieve a higher throughput that is required for a newborn screening laboratory, 4 analyzers are grouped together into a workstation for a throughput of up to

Conflict of interest

None.

Acknowledgments

We acknowledge Dr. Shu Chaing (NCDPH) for providing de-identified normal DBS, Duke University Biochemical Genetics Laboratory, Shire Human Genetic Therapies, Inc., and the Centers for Disease Control and Prevention for providing affected DBS, and Donovan Bort at Advanced Liquid Logic Inc. for technical assistance. Research reported in this publication was partly supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health

References (26)

  • D.S. Millington et al.

    Tandem mass spectrometry: a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism

    J Inherit Metab Dis

    (1990)
  • O. Staretz-Chacham et al.

    Lysosomal storage disorders in the newborn

    Pediatrics

    (2009)
  • D.S. Millington

    Rapid and effective screening for lysosomal storage disease: how close are we?

    Clin Chem

    (2008)
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