High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening

UMMS Affiliation

Department of Pediatrics; New England Newborn Screening Program

Publication Date


Document Type



Blood Specimen Collection; Calibration; DNA; Feasibility Studies; Gene Dosage; Genes, T-Cell Receptor; Humans; Infant, Newborn; Intensive Care Units; *Neonatal Screening; Polymerase Chain Reaction; Quality Control; Receptors, Antigen, T-Cell; Regression Analysis; Ribonucleases; Severe Combined Immunodeficiency


Genetics and Genomics | Medical Genetics | Pediatrics


BACKGROUND: Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample.

METHODS: We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded.

RESULTS: The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR.

CONCLUSIONS: Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

DOI of Published Version



Clin Chem. 2010 Sep;56(9):1466-74. Epub 2010 Jul 21. Link to article on publisher's site

Journal/Book/Conference Title

Clinical chemistry

Related Resources

Link to Article in PubMed

PubMed ID