Abstract |
Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS.
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Authors | Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S Abraham, Devin Oglesbee |
Journal | The Journal of molecular diagnostics : JMD
(J Mol Diagn)
Vol. 19
Issue 5
Pg. 755-765
(09 2017)
ISSN: 1943-7811 [Electronic] United States |
PMID | 28826609
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. |
Chemical References |
- Genetic Markers
- Receptors, Antigen, T-Cell
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Topics |
- Case-Control Studies
- Female
- Genetic Markers
- Humans
- Infant
- Infant, Newborn
- Lymphocytes
(metabolism)
- Male
- Multiplex Polymerase Chain Reaction
- Neonatal Screening
(methods)
- Real-Time Polymerase Chain Reaction
(methods)
- Receptors, Antigen, T-Cell
(genetics)
- Reproducibility of Results
- Sensitivity and Specificity
- Severe Combined Immunodeficiency
(diagnosis, genetics)
- Workflow
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