Study Design for an Evaluation of Newborn Screening for SCID in the UK

Severe combined immunodeficiency is a rare inherited disorder, which, if untreated, invariably proves fatal in late infancy or early childhood. With treatment, the prognosis is much improved. Early treatment of the siblings of cases, before they become symptomatic, has shown considerable improvements in outcomes. Based on this and the development of a test that can be used on the whole population of neonates (measurement of T-cell receptor excision circles—TRECs), many countries have added it to their routine newborn bloodspot screening programmes. The UK National Screening Committee (UKNSC) has considered whether SCID should be added to the UK screening programme and concluded that it was likely to be cost effective, but that there were a number of uncertainties that should be resolved before a national roll-out could be recommended. These include some aspects of the test, such as: cost; the use of different assays and cut-off levels to reduce false positive rates, while maintaining sensitivity; the overall benefits of screening for disease outcome in patients with SCID and other identified disorders; the need for a separate pathway for premature babies; the acceptability of the screening programme to parents of babies who have normal and abnormal (both true and false positive) screening results. To achieve this, screening of two thirds of babies born in England over a two-year period has been planned, beginning in September 2021. The outcomes and costs of care of babies identified by the screening will be compared with those of babies identified with SCID in the rest of the UK. The effect of the screening programme on parents will form part of a separate research project.

Neonatal abstinence syndrome is a potential cause of low TREC copy number

Severe combined immunodeficiency (SCID) is a rare genetic condition characterized by significant T cell lymphopenia and impaired T cell function. Many jurisdictions use the quantitation of T cell receptor excision circles (TRECs) to screen for SCID in newborns, but false positives may be seen in several conditions. We report 3 newborns with neonatal abstinence syndrome who presented with decreased TREC copy number.

Establishing Newborn Screening for SCID in the USA; Experience in California

Newborn screening for severe combined immunodeficiency (SCID) has developed from the realization that infants affected with SCID require prompt diagnosis and treatment to avoid fatal infectious complications. Screening DNA from infant dried blood spots for T-cell receptor excision circles (TRECs), byproducts of normal antigen-receptor gene rearrangement, has proven to be a reliable method to identify infants with SCID and other serious T lymphocyte defects before the onset of serious infections. The experience of the SCID newborn screening program in California after screening over 3 million infants demonstrates the effectiveness of this measure.

First Year of TREC-Based National SCID Screening in Sweden

Screening for severe combined immunodeficiency (SCID) was introduced into the Swedish newborn screening program in August 2019 and here we report the results of the first year. T cell receptor excision circles (TRECs), kappa-deleting element excision circles (KRECs), and actin beta (ACTB) levels were quantitated by multiplex qPCR from dried blood spots (DBS) of 115,786 newborns and children up to two years of age, as an approximation of the number of recently formed T and B cells and sample quality, respectively. Based on low TREC levels, 73 children were referred for clinical assessment which led to the diagnosis of T cell lymphopenia in 21 children. Of these, three were diagnosed with SCID. The screening performance for SCID as the outcome was sensitivity 100%, specificity 99.94%, positive predictive value (PPV) 4.11%, and negative predictive value (NPV) 100%. For the outcome T cell lymphopenia, PPV was 28.77%, and specificity was 99.95%. Based on the first year of screening, the incidence of SCID in the Swedish population was estimated to be 1:38,500 newborns.

Investigating the Variation of TREC/KREC in Combined Immunodeficiencies

T-cell receptor excision circles (TREC)/Kappa-deleting recombination excision circles (KREC) assay has been recently recognized for detecting patients with primary (T- and/or B-cell) immunodeficiency (PID). We aimed to investigate the alterations of these biomarkers in some combined immunodeficiency patients compared to the healthy controls in different age groups. TREC and KREC were assessed in a total of 82 PID patients, most of them with exact genetic diagnosis (3 months to 42 years); using quantitative real-time-polymerase chain reaction (PCR). Patients had a final diagnosis of common variable immunodeficiency (n=23), ataxia-telangiectasia (AT) (n=17), hyper-IgE syndrome (HIES) (7 with DOCK8 deficiency, 4 with signal transducer and activator of transcription 3 (STAT3) deficiency, and 8 children with unknown genetic defects), Wiskott-Aldrich syndrome (WAS) (n=20), purine nucleoside phosphorylase (PNP)deficiency(n=1), dedicator of cytokinesis2 (DOCK2) deficiency (n=1), recombinase activating gene1 (RAG1) deficiency (n=1). Very low to zero amounts of TREC and/or KREC were detected in 14 out of 23 cases of common variable immunodeficiency (CVID), 14 out of 17 cases of AT, 8 out of 20 cases of WAS, 6 out of 7 cases of DOCK8-deficiency patients, 4 out of 8 cases of HIES with unknown genetic defects and all patients with defects in DOCK2, PNP, and RAG1. STAT3-deficient patients were normal for both biomarkers. All patients showed a significant difference in both markers compared to age-matched healthy controls. Our findings highlight that apart from severe types of T/B cell defects, this assay can also be used for early diagnosis the patients with late-onset of disease and even PIDs without a positive family history.

Newborn Screening for Severe Combined Immunodeficiency Using the Multiple of the Median Values of T-Cell Receptor Excision Circles

All newborn screening programs screen for severe combined immunodeficiency by measurement of T-cell receptor excision circles (TRECs). Herein, we report our experience of reporting TREC assay results as multiple of the median (MoM) rather than using conventional copy numbers. This modification simplifies the assay by eliminating the need for standards with known TREC copy numbers. Furthermore, since MoM is a measure of how far an individual test result deviates from the median, it allows normalization of TREC assay data from different laboratories, so that individual test results can be compared regardless of the particular method, assay, or reagents used.

Neonatal Lymphopenia Screening Is Important For Early Diagnosis of Severe Combined Immunodeficiency

Objective T-cell receptor excision circles are expensive for neonatal severe combined immunodeficiency screening in developing countries. We aimed to detect immunodeficiencies presenting with lymphopenia to enable screening in the general population and to improve awareness regarding lymphopenia among clinicians. Study Design This study was conducted prospectively. In all newborns included, complete blood count from umbilical cord blood samples was recorded. Absolute lymphopenia was defined as absolute lymphocyte count <3,000/mm3 in umbilical cord blood sample. Complete blood count was repeated at month 1 in cases found to have lymphopenia. Results Overall, 2,000 newborns were included in the study. Absolute lymphopenia was detected in 42 newborns (2.1%), while lymphocyte count was >3,000/mm3 in 1,958 newborns (97.9%). Two infants with persisted lymphopenia at the end of the first month; therefore, further evaluations such as lymphocyte subsets for severe combined immunodeficiency (SCID) were done. In the first infant, the lymphocyte subgroups were detected as compatible with T (−), B (−), natural killer cells (NK) (+) SCID phenotype RAG defect. Sanger sequencing revealed that NM_000448 c.2209C > T (p.R737C) homozygous mutation of RAG1 gene. In the other infant, the lymphocyte subgroups were found as considered with T (−), B (+) NK (−) SCID phenotype JAK3 defect. Both patients underwent hematopoietic stem cell transplantation from human leukocyte antigen-matched family member. Conclusion Absolute lymphopenia by complete blood count is a more simpler, relatively noninvasive and inexpensive screening methodfor detection of SCID in newborns compared with T-cell receptor excision circles technique. Key Points

Toward the Analysis of Lymphocyte Development in Space: PCR-Based Amplification of T-Cell Receptor Excision Circles (TRECs) Aboard the International Space Station

Spaceflight offers vast possibilities for expanding human exploration, whereas it also bears unique health risks. One of these risks is immune dysfunction, which can result in the reactivation of latent pathogens and increased susceptibility to infections. The ability to monitor the function of the immune system is critical for planning successful long-term space travel. T lymphocytes are immune cells that develop in the thymus and circulate in the blood. They can detect foreign, infected, or cancerous cells through T cell receptors (TCRs). The assembly of TCR gene segments, to produce functional TCR genes, can be monitored by measuring the presence of TCR excision circles (TRECs), circular fragments of DNA that are by-products of this assembly process mediated by the V(D)J recombination machinery. In this study, we used polymerase chain reaction (PCR) on the International Space Station (ISS) to detect TRECs in murine peripheral blood. We were able to detect TRECs in the blood of normal healthy mice of different ages, with an efficiency comparable to that achieved in ground controls. As expected, we were unable to detect TRECs in the blood of immunodeficient mice. These results are the first step in optimizing a specific, rapid, safe, and cost-effective PCR-based assay to measure the integrity of mammalian immune systems during spaceflight.