A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar.

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A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S. Abraham, Devin Oglesbee The Journal of Molecular Diagnostics Volume 19, Issue 5, Pages 755-765 (September 2017) DOI: 10.1016/j.jmoldx.2017.05.011 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Workflow of TREC ddPCR analysis. Isolated genomic DNA from DBSs is added to a PCR and partitioned into droplets by the AutoDG. After end-point amplification, droplets are singularized by the QX200 reader, and fluorescence is measured as FAM and HEX signal amplitudes. Pink lines indicate FAM and HEX signal amplitude thresholds applied to the analysis for designating positive or negative droplets. The Journal of Molecular Diagnostics 2017 19, 755-765DOI: (10.1016/j.jmoldx.2017.05.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 The clustering of droplets in the ddPCR analysis shown as a heat map. Larger numbers of droplets are represented in red. Droplet cloud thresholds are shown as pink lines. A: Negative droplets (NEG; absent TREC or RPP30 PCR products) are shown; blue represents positive droplets containing TREC PCR product, RPP30 PCR product, or double-positive droplets with both TREC and RPP30 PCR products. B: Lack of TREC-positive droplets is seen for an SCID-positive infant. C: Lack of TREC- or RPP30-positive droplets is seen for a sample without template DNA. D: The level of ddPCR carryover observed for a blank paper specimen run after a DBS with our NBS process. Pink lines indicate FAM and HEX signal amplitude thresholds applied to the analysis for designating positive or negative droplets. The Journal of Molecular Diagnostics 2017 19, 755-765DOI: (10.1016/j.jmoldx.2017.05.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Distribution of population-based reference ranges and affected cases. A and B: Dot plots show the range of TREC (A) and RPP30 (B) levels in the normal reference range study and for low TREC patients. Data plotted as log10. Dashed lines represent the TREC and RPP30 assay thresholds. C and D: Histograms illustrating the distribution of TREC and RPP30 copies in full-term infants observed during the population-based study. The Journal of Molecular Diagnostics 2017 19, 755-765DOI: (10.1016/j.jmoldx.2017.05.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 The proposed SCID newborn screening algorithm with ddPCR. CD4RT, CD4 T-cell recent thymic emigrant; NICU, neonatal intensive care unit; TBBS, T- and B-cell quantification by flow cytometry; TREC, B, T-cell receptor excision circles analysis, blood. The Journal of Molecular Diagnostics 2017 19, 755-765DOI: (10.1016/j.jmoldx.2017.05.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions