Assessment of Reader Technologies for Over-the-Counter Diagnostic Testing.

Background: Over-the-counter (OTC) diagnostic testing is on the rise with many in vitro diagnostic tests being lateral flow assays (LFAs). A growing number of these are adopting reader technologies, which provides an alternative to visual readouts for results interpretation, allowing for improved accessibility of OTC diagnostics. As the reader technology market develops, there are many technologies entering the market, but no clear, single solution has yet been identified. The purpose of this research is to identify and discuss important parameters for the assessment of LFA reader technologies for consideration by manufacturers or researchers. Methods: As part of The National Institute of Biomedical Imaging and Bioengineering's Rapid Acceleration of Diagnostics (RADx) Tech program, reader manufacturers were interviewed to investigate the current state of reader technology development through several parameters identified as important industry standards. Readers were categorized by technology type and parameters including cost, detection method, multiplex capabilities, assay type, maturity, and use case were all assessed. Results: Fifteen reader manufacturers were identified and interviewed, and information on a total of 19 technologies was assessed. Reader technology type was found to be predictive of other attributes, whether the reader is smart technology only, a standalone reader, a reader with smart technology required, or a reader with smart technology optional. Conclusions: Pairing reader technology with OTC diagnostic tests is important for improving existing COVID-19 tests and can be utilized in other diagnostics as the OTC use case grows in popularity. Reader technology type, which is predictive of core reader attributes, should be considered when selecting a reader technology for a specific LFA test within the context of regulatory guidance. As diagnostics increase in complexity, readers provide solutions to accessibility challenges, facilitate public health reporting, and ease the transition to multiplex testing, therefore increasing market availability.

Dataset and standard operating procedure for newborn screening of six lysosomal storage diseases: By tandem mass spectrometry.

In this data article we provide a detailed standard operating procedure for performing a tandem mass spectrometry, multiplex assay of 6 lysosomal enzymes for newborn screening of the lysosomal storage diseases Mucopolysaccharidosis-I, Pompe, Fabry, Niemann-Pick-A/B, Gaucher, and Krabbe, (Elliott, et al., 2016) [1]. We also provide the mass spectrometry peak areas for the product and internal standard ions typically observed with a dried blood spot punch from a random newborn, and we provide the daily variation of the daily mean activities for all 6 enzymes.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BACKGROUND: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. OBJECTIVE: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. METHODS AND RESULTS: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). DISCUSSION: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Validation of a robust PCR-based assay for quantifying fragile X CGG repeats.

BACKGROUND: Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats. METHODS: We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed. RESULTS: All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV<2.5%) and inter-assay imprecision was within 1 CGG repeat. CONCLUSION: This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.

A novel assay for evaluating fragile X locus repeats.

We have developed a novel fragile X locus repeat assay that is a simple and high-throughput method that, with clinical validation, may be suitable for screening. It uses amplification of the FMR1 trinucleotide repeat region, followed by a hybridization assay to quantify the number of repeats in the amplicons. To our knowledge, this is the first repeat-counting assay that uses fluorescent signals rather than electrophoresis or mass spectrometry as the signaling mechanism. We also report the development of a simple microfluidic electrophoresis reflex test that uses the same amplicons and reduces the need for Southern blots to differentiate homozygous female normal samples from full mutations. The new assay, which is based on a suspension-array hybridization method, was tested on a series of male and female reference samples spanning the range from normal to full mutations. It was also tested on DNA from 1008 dried blood spot samples from pregnant women in their first trimester. The hybridization assay identified 51 of those as potentially expanded alleles of ≥45 repeats or as intermediate or higher in FMR1 repeat classification. Of these screen-positive samples, eight were confirmed by microfluidic electrophoresis as premutations consisting of ≥55 repeats. The FMR1 repeat assay is straightforward to run in high throughput, and the results are in the form of numerical ratios for ease of initial interpretation.

The development of a rapid assay for prenatal testing of common aneuploidies and microdeletion syndromes.

OBJECTIVE: To develop a novel, rapid prenatal assay for pregnancies with high likelihood of normal karyotypes, using BACs-on-Beads(™) technology, a suspension array-based multiplex assay that employs Luminex(®) xMAP(®) technology, for the detection of gains and losses in chromosomal DNA. METHODS: Fifteen relatively common microdeletions were selected that are not detectable, or may be missed, by karyotyping and usually do not present with abnormal ultrasound findings. Chromosomes 13, 18, 21, X, and Y were included. We validated the assay with 430 samples. RESULTS: All microdeletions and aneuploidies were correctly identified, except for a 69,XXX incorrectly identified as a normal female and a male with ∼20% maternal cell contamination (MCC) that could not be distinguished from 69,XXY. MCC became apparent at 20 to 30%. Mosaicism was identified at 30 to 35% abnormal cells. CONCLUSION: We have developed an alternative to fluorescence in situ hybridization (FISH) aneuploidy screening and microarray analysis in otherwise normal pregnancies undergoing invasive testing. We demonstrated that the assay will detect all microdeletions and aneuploidies of regions covered on the assay. We developed analytical software that displays results for well-characterized syndromes but not abnormalities of unclear clinical significance. This assay is likely to be preferred by women seeking testing beyond routine karyotyping but who desire more information than provided by aneuploidy FISH.