Development of a routine newborn screening protocol for severe combined immunodeficiency

doi:10.1016/j.jaci.2009.04.007

Journal of Allergy and Clinical Immunology

Volume 124, Issue 3, September 2009, Pages 522-527

https://doi.org/10.1016/j.jaci.2009.04.007 Get rights and content

Background

Severe combined immunodeficiency (SCID) is characterized by the absence of functional T cells and B cells. Without early diagnosis and treatment, infants with SCID die from severe infections within the first year of life.

Objective

To determined the feasibility of detecting SCID in newborns by quantitating T-cell receptor excision circles (TRECs) from dried blood spots (DBSs) on newborn screening (NBS) cards.

Methods

DNA was extracted from DBSs on deidentified NBS cards, and real-time quantitative PCR (RT-qPCR) was used to determine the number of TRECs. Positive controls consisted of DBS from a 1-week-old T⁻B⁻NK⁺ patient with SCID and whole blood specimens selectively depleted of naive T cells.

Results

The mean and median numbers of TRECs from 5766 deidentified DBSs were 827 and 708, respectively, per 3.2-mm punch (∼3 μL whole blood). Ten samples failed to amplify TRECs on initial analysis; all but 1 demonstrated normal TRECs and β-actin amplification on retesting. No TRECs were detected in either the SCID or naive T-cell–depleted samples, despite the presence of normal levels of β-actin.

Conclusions

The use of RT-qPCR to quantitate TRECs from DNA extracted from newborn DBSs is a highly sensitive and specific screening test for SCID. This assay is currently being used in Wisconsin for routine screening infants for SCID.

Section snippets

Institutional review and approval

The protocol to use deidentified NBS specimen collection cards from the Wisconsin State NBS program was approved by the University of Wisconsin Health Sciences Institutional Review Board.

Screened samples

Deidentified DBSs used for this assay development were punched from NBS cards submitted to the Wisconsin State Laboratory of Hygiene NBS Laboratory. Blank filter paper NBS collection cards spotted with peripheral blood from a T^–B⁻NK⁺ patient with SCID (pre-HSCT) were used as a positive control for the TREC

Results

The first step of the SCID assay protocol is to isolate the DNA optimally from 3.2-mm DBSs for RT-qPCR. To accommodate the need for a high-throughput and yet inexpensive procedure for a NBS program, we used commercially available reagents (see Methods) to extract DNA from DBS. DNA extraction methodology and the RT-qPCR for TRECs were initially optimized by using DBS from 4020 deidentified full-term infants (≥37 weeks of gestation, and data not shown). Fig 1 demonstrates an example of a RT-qPCR

Discussion

We have successfully optimized the method of measuring TRECs from NBS DBSs by enhancing the yield of DNA extraction and increasing the amplification efficiency of the RT-qPCR. In addition, by retesting NBS cards that were low for TRECs on our initial screening test, we were able to reduce markedly the screening positive results using this protocol. The TREC assay is reliable, reproducible, inexpensive, and easy to use. Importantly, the TREC assay is highly sensitive for detecting abnormally low

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: Supported by the Jeffrey Modell Foundation, the Children's Hospital of Wisconsin Foundation, the Children's Research Foundation, and the Wisconsin State Laboratory of Hygiene.

: Disclosure of potential conflict of interest: M. W. Baker has received research support from the Jeffrey Modell Foundation and the Centers for Disease Control and Prevention. W. J. Grossman is an employee of Merck & Co and has received research support from the National Institutes of Health and the Midwest Athletes Against Childhood Cancer (MACC) Fund. R. H. Laessig has received research support from the Jeffrey Modell Foundation and has provided legal consultation or expert witness testimony in a case related to methodology. J. M. Routes has received research support from the National Institutes of Health and Baxter Pharmaceuticals. The rest of the authors have declared that they have no conflict of interest.

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Immune deficiencies, infection, and systemic immune disordersDevelopment of a routine newborn screening protocol for severe combined immunodeficiency

Background

Objective

Methods

Results

Conclusions

Section snippets

Institutional review and approval

Screened samples

Results

Discussion

Pathology

J Allergy Clin Immunol

J Allergy Clin Immunol

Mol Genet Metab

J Pediatr

J Allergy Clin Immunol

Lancet

J Allergy Clin Immunol

Immune deficiencies, infection, and systemic immune disorders
Development of a routine newborn screening protocol for severe combined immunodeficiency