Efficient screening strategies for severe combined immunodeficiencies in newborns

ABSTRACT Introduction Severe combined immunodeficiency (SCID) is one of the most severe forms of inborn errors of immunity (IEI), affecting both cellular and humoral immunity. Without curative treatment such as hematopoietic stem cell transplantation or gene therapy, affected infants die within the first year of life. Due to the severity of the disease, asymptomatic status early in life, and improved survival in the absence of pretransplant infections, SCID was considered a suitable candidate for newborn screening (NBS). Areas covered Many countries have introduced SCID screening based on T-cell receptor excision circle (TREC) detection in their NBS programs. Screening an entire population is a radical departure from previous paradigms in the field of immunology. Efficient screening strategies are cost-efficient and balance high sensitivity while preventing high numbers of referrals. NBS for SCID is accompanied by (actionable) secondary findings, but many NBS programs have optimized their screening strategy by adjusting algorithms or including second-tier tests. Harmonization of screening terminology is of great importance for international shared learning. Expert Opinion The expansion of NBS is driven by the development of new test modalities and treatment options. In the near future, other techniques such as next-generation sequencing will pave the way for NBS of other IEI. Exciting times await for population-based screening programs.


Introduction
Severe combined immunodeficiency (SCID) is one of the most severe forms of inborn errors of immunity (IEI). The phenotype is characterized by the absence or dysfunction of T-lymphocytes often accompanied by the lack of functional B-lymphocytes and NK-cells affecting both cellular and humoral immunity [1]. SCID is a term used to describe a disease entity caused by various genetic defects [2]. Without adaptive immunity, patients with SCID are prone to severe, recurrent infections caused by both non-opportunistic and opportunistic pathogens. Patients are usually born asymptomatic but develop life-threatening infections and failure to thrive in the first months of life. Without curative treatment, in the form of allogeneic hematopoietic stem cell transplantation (HSCT) or in some specific forms of SCID, gene therapy (GT), affected infants die within the first year of life [3][4][5].
The importance of an early diagnosis is demonstrated by studies showing improved survival of SCID patients diagnosed at birth due to a positive family history (OS 85-90%) compared to the first presenting family members (OS 40-42%) [6,7]. These observed differences were irrespective of conditioning regimen, donor source, or underlying (genetic) diagnosis suggesting the relation between improved survival and early diagnosis. In addition, retrospective multi-center studies in larger SCID patients' cohorts have shown that patient outcomes are significantly improved when curative therapy with HSCT is performed before the age of 3.5 months and/or prior to the onset of severe and debilitating infections [8]. Survival rates were adversely impacted by active infection pretransplantation; 81% for patients with active infection at the time of transplantation versus 95% in infection-free patients [9]. These findings suggest that an early diagnosis and the prevention of infections are predominant determinants of a good transplantation outcome. Many SCID cases are sporadic, with no positive family history and have nonspecific disease manifestations leading to delay in recognition of the underlying disease and subsequently to delay in treatment. Realistically, an early diagnosis prior to the development of life-threatening complications is only achievable by early identification of infants with SCID through newborn screening (NBS) programs.

Differences between diagnostics and screening
In the past decade, NBS for SCID has been introduced in many screening programs worldwide. SCID is the first IEI to be accepted for population-based screening, and implementation has provided important clinical benefits for affected infants. Screening an entire population for these types of conditions is a radical departure from previous scientific and medical paradigms in the field of immunology. Instead of being centered on a patient with signs and symptoms of disease, NBS applies a simple assay for a biomarker to every infant in a population CONTACT Maartje Blom m.blom@lumc.nl Laboratory for Pediatric Immunology, Willem-Alexander Children's Hospital, Leiden University Medical Center, Leiden 2300 RC, The Netherlands of whom the vast majority are not affected [10]. There are clear differences between screening and diagnostics. The primary purpose of screening is to detect early disease in a large number of apparently healthy individuals. Most screening tests are not designed to establish a diagnosis, but rather to signal the potential for a serious condition for which specific follow-up must be initiated. Diagnostic testing, on the other hand, is performed to establish the presence (or absence) of a disease in symptomatic or screen positive individuals (confirmatory test) [11]. Efficient screening strategies should be acceptable to the general population and have a (non)invasive character, and benefits should justify the costs (Figure 1).

What makes a screening strategy efficient?
Public health programs have the responsibility to continuously optimize screening programs for their stakeholders pursuing the most efficient screening strategy that balances high sensitivity while preventing high numbers of referrals. However, what defines an efficient screening strategy for SCID, what could be improved in current screening strategies and what lies ahead in the future? The primary aim of NBS programs is to identify potentially fatal or disabling conditions in presymptomatic newborns for which timely intervention is available and critical to improve the outcome. These conditions might not be evident at birth, but if left undiagnosed and untreated could have fatal or severe developmental consequences for the child. With early detection and early intervention, morbidity and mortality can be reduced. In addition to individual health benefits, NBS also aims to minimize negative societal and economic impacts of life-threatening diseases [12].
The recommendations of Wilson and Jungner (1968) for populated-based disease screening are the backbone of the screening policy [13]. Since their publication in 1968, these criteria have provided a framework against which conditions can be assessed for their suitability for screening, being of aid in decision making with regard to inclusion of new disease candidates in NBS programs. The criteria have been refined in 2008 by the WHO due to the growing interest in genetic screening and changing demands of modern times (Table 1) [16]. Efficient screening strategies take these predefined criteria into account.
An efficient screening test has to be extremely sensitive, as missing patients due to false-negative results defeat the purpose of detecting all affected cases. In addition, as trust in population screening programs is one of the key elements for parents to participate in NBS, NBS programs aim for the highest sensitivity to avoid missing affected children in the direct health interest of the child. At the same time, screening tests must be highly specific to avoid the referral of a large percentage of the general population for diagnostic testing. From the perspective of healthcare utilization, downstream referral centers should not be overrun with falsepositive cases and excessive cost. Public health programs have a responsibility toward the society as a whole, as screening requires resources, and referrals are associated with high diagnostic costs. In addition, referrals are associated with high emotional impact for parents and invasive diagnostic testing for the child [17][18][19]. Some false-positive cases are considered an acceptable tradeoff to avoid falsenegatives; however, many NBS programs believe that

Article highlights
• Newborn screening (NBS) for severe combined immunodeficiency (SCID) is based on the detection of T-cell receptor excision circles (TRECs). • The primary purpose of screening is early detection of the condition in a large number of apparently healthy individuals, while diagnostics are performed to establish the presence (or absence) of a disease in symptomatic or screen positive individuals. • Efficient screening strategies are cost-efficient and pursue high sensitivity while preventing high numbers of false positive referrals. • NBS for SCID is accompanied by (actionable) secondary findings, but many NBS programs have optimized their screening strategy by adjusting algorithms or including second-tier tests • Harmonization of screening terminology is of great importance for international shared learning. • Expansion of NBS is driven by the development of new test modalities and treatment options. • Other techniques such as epigenetic immune cell counting and nextgeneration sequencing will path the way for NBS of other inborn errors of immunity. • Ethical, social, and legal implications, logistics, and costs will have to be carefully considered before different IEI can be considered as suitable candidates for NBS programs. secondary findings in a program aimed at screening should be avoided where possible. It is recommended to consistently opt for a test method with the lowest chance of secondary findings, provided multiple tests are available.

History of screening strategies for SCID
Several screening strategies have been proposed to identify patients with SCID directly after birth. A complete blood count (CBC) was suggested to detect T-cell lymphopenia, but this simple laboratory test lacked sensitivity as patients with present B-lymphocytes, maternal engraftment, or oligoclonal expansion would be missed [20]. The same was the case for protein immunoassays on dried blood spots for T-cell specific markers such as CD3 [21]. Subsequently, flow cytometry to determine T-cell populations in cord blood was also considered, but proved to be too time-consuming and expensive for a population screening test [22]. There was a need for an extremely sensitive and specific biomarker that could identify T-cell lymphopenia shortly after birth, while avoiding excessive costs and anxiety associated with false-positive screen results [23].

The golden standard: T-cell receptor excision circles (TRECs)
V(D)J recombination of the TCR loci is the process whereby a diverse repertoire of antigen receptors is generated. In each T-cell randomly chosen combinations of variable (V), diversity (D) and joining (J) segments are formed to synthesize a unique rearrangement in each cell. Only T-cell progenitors with inframe rearranged locus are selected to survive and mature. The excised DNA fragments that are not destined to be incorporated into the mature TCR locus can be joined at their ends to form a great variety of circular DNA byproducts, called T-cell receptor excision circles (TRECs). During the δREC-ψJα rearrangement in the TCRA locus, the TCRD gene is excised and the δRec-ψJα signal joint TREC is formed. It is estimated that 70-80% of the thymocytes that ultimately express αβ TCR form a specific circular DNA TREC in this process ( Figure 2) [24,25]. Quantitative PCR amplification across the joined ends of the δRec-ψJα TREC reflects the number of recently formed T-cells in peripheral blood. TRECs were found to be unique to naïve αβ T-cells as non-naïve T-cells lack the δRec-ψJα signal joint. In addition, TRECs were considered to be an ideal marker for naïve T-cell production as they were noted to be stable and remained extrachromosomal in the cytoplasm of the T-cells, not replicating during mitosis. As a result, TRECs become diluted when the T-cell population expands through cell division [26]. In 2005, the first application of quantitative PCR for TREC detection as a large-scale population screening method for SCID was described [27]. With now a suitable and acceptable test method, an asymptomatic phase early in life and a definitive treatment before the onset of infections resulting in the best outcomes, important Wilson and Jungner screening criteria were met.
SCID became the first immune disorder in the NBS program and at the same time the TREC assay became the first highthroughput DNA-based NBS test. Since the introduction of TREC-screening, more SCID patients have been diagnosed than the previous estimated incidence rate based on retrospective analyses [28] This suggests the detection of patients who would have died due to severe infections prior to a diagnosis without NBS. In addition, recent studies show that the overall survival is significantly higher for newborns identified via NBS in comparison to children identified via critical illness or family history [29].

Secondary findings in NBS for SCID
Every population screening program has to deal with secondary or incidental findings: screen positive findings that are not the target disease and are not intended by the primary aim of the program. Low TREC levels indicate that a T-cell developmental problem might be present, but referral to the pediatric-immunologist is needed to confirm T-cell lymphopenia and to identify the underlying cause [30,31]. Even though There should be quality assurance, with mechanisms to minimize potential risks of screening 7. The natural history of the condition, including development from latent to declared disease, should be adequately understood 7. The program should ensure informed choice, confidentiality and respect for autonomy 8. There should be an agreed policy on whom to treat as patients 8. The program should promote equity and access to screening for the entire target population 9. The cost of case-finding (including diagnosis and treatment of patients diagnosed) should be economically balanced in relation to possible expenditure on medical care as a whole 9. Program evaluation should be planned from the outset 10. Case-finding should be a continuing process and not a 'once and for all' project 10. The overall benefits of screening should outweigh the harm TRECs are a highly sensitive biomarker for T-cell lymphopenia, they are nonspecific markers for the primary target disease SCID, introducing the field of NBS to a palette of neonatal conditions and disorders associated with low T-cells around birth. Low TREC levels can be identified in other forms of IEI such as less profound combined immunodeficiencies classified by the IUIS [2]. Newborns with a recognized genetic syndrome that includes low T-cell numbers within its spectrum of clinical findings can also present with low TREC numbers. Examples are newborns with 22q11.2 deletion syndrome (DiGeorge syndrome), CHARGE-syndrome, trisomy 21, ataxia telangiectasia, trisomy 18, and Jacobsen syndrome. TRECs and T-cell numbers can also be reversibly reduced due to secondary causes such as congenital malformations (e.g. cardiac or gastrointestinal anomalies), or disease processes without an intrinsic defect in the production of circulating cells (e.g. loss into third space in hydrops or chylothorax or vascular leakages in sepsis) [28,32]. Maternal immunosuppressant use can also be a cause of transient neonatal T-cell lymphopenia [33][34][35]. In these cases, T-cell lymphopenia usually resolves once excess T-cell losses or suppression of T-cell maturation has been abrogated. In newborns with idiopathic T-cell lymphopenia, TRECs, and T-cells might be low without an identified underlying cause, even after immunologic and comprehensive genetic evaluation. For infants with T-cell lymphopenia, longitudinal immunological evaluation is important to determine if the T-cell lymphopenia is transient [36]. Infants with preterm birth (gestational age <37 weeks) and/or low birth weight are a disproportionate source of abnormal TREC results, and many screening programs have adjusted their screening algorithm for these cases by including repeated sampling. T-cell lymphopenia in these infants is depending on the degree of thymic maturity, although T-cells are not functionally impaired, and T-cell numbers usually normalize with increasing gestational age [37].
Finally, in the case of an abnormal TREC value, but normal levels of T-cells (>1500/μL and > 200 naïve/μl) no further immunological work-up is required within the SCID screening context [38]. In these cases, TRECs could have been low at the time of the heel prick due to transient T-cell lymphopenia that resolved in the first weeks up to referral. Enumeration of naïve T-cells should always be included into follow-up to exclude rare cases of maternal engraftment or oligoclonal expansion. TRECs could also be low due to technical test errors leading to false-positive results. Uniform follow-up protocols are required for a prompt and consistent approach to a definitive diagnosis and can provide guidance for pediatrician-immunologists when dealing with these non-SCID cases identified via NBS for SCID.
Prior to implementation of the TREC-assay, it was already suspected that previously unrecognized conditions would come to light with screening. Neonatal T-cell lymphopenia and low TRECs in trisomy 21, CLOVES syndrome, and RAC2 were not associated with neonatal lymphopenia prior to NBS for SCID. Variants in new genes such as BCL11B and EXTL3 were new T-cell deficiencies discovered with whole exome sequencing (WES) and functional analysis after an abnormal TREC result [10]. More data will become available on the management of secondary findings in the next years as more countries are adopting SCID screening in their NBS programs. It would therefore be recommended for NBS program to not only register follow-up data of patients with the target disease, but also include outcome data of patients with other causes for low TRECs.

Actionable secondary findings in NBS for SCID
A distinction can be made between three categories of secondary findings: 1) clinically relevant findings; 2) clinical findings (as yet) unclear; and 3) findings that are not clinically relevant. Within the category of clinically relevant findings, further distinction can be made between actionable findings where treatment or prevention is possible, and non-actionable findings that may be relevant prognostically, but for which no Figure 2. Trecs are stable, circular fragments of DNA formed during by excisional rearrangements of the TCR genes. During the δREC-ψJα rearrangement in the TCRA locus, the TCRD gene is excised and the δRec-ψJα signal joint TREC is formed. The δRec-ψJα coding joint might still be present on the nonfunctional TCRAD allele and by subsequent Vα-Jα rearrangements, the δREC-ψJα coding joint will be removed and placed on a novel excision circle. With quantitative PCR amplification across the joined ends of the δRec-ψJα TREC the number of recently formed T-cells in peripheral blood can be determined. treatment or prevention is available. It can be difficult to make clear statements about actionability.
The term actionable in NBS for SCID can be linked to many of the secondary findings. Neonates with profound T-cell lymphopenia, not meeting all criteria for SCID but eligible for HSCT, would undisputedly be classified as actionable findings. The same would be applicable for patients with complete 22q11.2 deletion syndrome (DiGeorge syndrome), CHARGE syndrome, or athymic FOXN1 deficiency, all of which are indications for thymus transplantation [39][40][41]. Cases of significant T-cell lymphopenia that might benefit from antibiotic prophylaxis, protective isolation, or avoiding live-attenuated vaccines should also be deemed actionable [10,38]. This is also the case for patients with A-T: the avoidance of ionizing radiation in diagnostic test, prevention of starting HSCT including a conditioning regime and avoidance life-attenuated vaccines are important to prevent malignancies and occurrence of serious infections by vaccine-strain organisms [42]. The term actionable is therefore more suitable than the term treatable, as withholding treatment can be an important early intervention leading to improved outcomes. Non-actionable secondary findings may be relevant prognostically, but either effective treatments are not available or health benefits from early diagnosis are limited or uncertain. The aim of population-based screening is to prevent morbidity or mortality from the targeted disorders through earlier treatment and with limited harm to unaffected infants. Non-actionable secondary findings and referrals of infants with normal lymphocyte numbers by flow cytometry raise concerns about the harm-benefit ratio of screening, and public health programs justifiably strive to prevent referral of these cases [18].
Some countries have included T-cell lymphopenia in addition to SCID as their primary or secondary target in their NBS program. Non-SCID T-cell lymphopenia cases would therefore no longer be considered as secondary findings to NBS for SCID. In the United States, SCID is the core condition, but T-cell related lymphocyte deficiencies are stated as secondary conditions: disorders that are recommended to be screened for while testing the core disorders on the Recommended Uniform Screening Panel (RUSP) [43]. In Norway, the primary target for NBS is SCID and other severe T-cell deficiencies [44]. In the Netherlands the new definition encompasses severe T-cell deficiencies for which HSCT, thymic transplantation, and gene therapy are indicated. Including the term T-cell deficiencies as a primary target poses some challenges as not all disorders with T-cell lymphopenia can be detected with the TREC assay. Combined immunodeficiencies such as ζ-associated protein of 70kDa (ZAP-70) deficiency or MHC class I and II gene expression deficiency, have severely impaired T-cell function but can have normal TREC levels as T-cell development is intact beyond the point of TCR gene recombination [45]. High sensitivity might therefore not always be achieved as newborns with T-cell deficiency can be missed with TREC screening. False-negative cases lower the trust in NBS program and vague target diseases diminish the clarity of the program as a whole. Parents might opt out of participation, whereas the program emphatically aims for a high level of participation to avoid missing very severe actionable conditions at birth. Adjusting a target definition in a NBS program would be accompanied with some changes. Clear information provision to parents is of utmost importance in this process. Disease-specific information should entail that SCID can be treated with HSCT, but actionable T-cell deficiencies can have other types of treatment. Genetic analysis in follow-up protocols should not only focus on variants for SCID, but also on identifying genetic defects for other actionable T-cell deficiencies in line with the IUIS classification [2]. Prior to genetic analysis, parents should be counseled by a clinical geneticist and pediatrician to inform them about severe disorders without highly effective treatment options such as A-T.

Adjustments in strategies for more efficient SCID screening
NBS programs use a variety of test methods, cutoff values, and screening algorithms to balance a high sensitivity, detecting all SCID patients, while preventing high referral rates in their particular populations. Selecting the correct cutoff value is of utmost importance for any screening test and should be validated in a larger cohort of samples. TREC cutoff values can differ between countries and are method-specific. Some programs have lowered their TREC cutoff values or adjusted their screening algorithms including urgent direct referrals and the request of a second NBS card [46][47][48][49][50][51][52]. Other programs have chosen to include a second tier test after initial TREC analysis such as next-generation sequencing (NGS) with gene panels [44,53]. Many NBS programs have incorporated adaptations in their screening algorithms for infants with preterm birth and/or low birthweight with low TREC levels to avoid high referral rates. In addition, other test methods such as tandem mass spectrometry, i.e. for ADA or PNP deficiency have been proposed as well [54,55]. There are other potential methods to reduce the number of secondary findings and false-positive referrals in NBS for SCID. Performing a second PCR with primers at different positions could prevent false-positive referrals caused by TREC region variations leading to primer/probe annealing problems and amplification failure [56,57]. Epigenetic immune cell counting as a second tier allows the measurement of relative (epi) CD3+ T-cells counts serving as more direct marker for absolute T-cells in comparison to TRECs [57,58]. Public health programs have a responsibility toward their stakeholders to continuously improve and optimize their NBS programs, therefore all possible adaptations leading to more targeted screening for the core condition and the reduction of false-positive referrals should be explored.

Is screening for SCID considered cost-efficient screening?
Many healthcare systems today are struggling with financial allocation and how much to invest in new medical products, services, and prevention and screening programs including ever-expanding NBS programs. The Wilson and Jungner criteria already stated in 1968 that the cost of case-finding should be economically balanced in relation to possible expenditure on medical care as a whole [13]. Decision analyses and economic evaluations including cost-effectiveness analyses can help inform policy decisions for NBS programs with regard to healthcare resource allocation. Several countries have performed economic evaluations for NBS for SCID, suggesting that NBS for SCID is cost-effective under a range of assumptions about incidence and costs [59][60][61][62][63][64]. Analysis based on reallife data resulted in higher costs, and consequently in less favorable cost-effectiveness estimates for NBS for SCID than previously published analyses based on hypothetical data [65]. Economic evaluations usually rely on model assumptions as information on long-term outcomes of NBS programs is scarce. Long-term outcomes are of key importance for defining the effectiveness of an intervention and should include outcome parameters such as increased incidence, (event-free) survival, morbidity and late complications, and quality of life (QoL) [66]. Accurate collection of data that address a broader societal perspective has been a known problem in economic evaluations. In addition to productivity costs, the perspective could be broadened further by including productivity loss of patients with a poor diagnosis or by including use of informal care. Health gain for patients with a non-SCID diagnosis identified via NBS for SCID could also lead to cost savings and more favorable cost-effectiveness results. Evaluation of QoL associated with a health state is complicated by methodological challenges such as the lack of validated methods for valuing QoL in young children and the need for proxy responders. There is a need for a largescale study on long-term outcomes of SCID patients after HSCT including societal factors such as QoL, productivity (school/work), and healthcare use included informal care.
The TREC assay is the first high-throughput DNA-based test in the NBS laboratory. It is important to keep in mind that introducing a new technique into a laboratory is associated with high cost for extra equipment, reagents, and personnel. Although NBS for SCID required introduction of DNA analysis as a primary screening modality for the first time, DNA technology as a platform in NBS laboratories is not limited to SCID. TRECs can be measured simultaneously with other biomarkers for disorders such as X-linked agammaglobulinemia (XLA) and spinal muscular atrophy (SMA) with multiplex PCR, allowing further expansion of NBS programs [67]. Costs for screening of one disorder would be significantly reduced if the test is adaptable to additional condition, as was observed for tandem mass-spectrometry. Even without multiplex PCRfor example, in NBS for congenital cytomegalovirus (CMV), the benefit of adaption of DNA extraction and qPCR technology would share costs for reagents, equipment, and personnel resulting in a more favorable cost-effectiveness ratio. In addition, if the primary target of NBS for SCID would be changed to 'SCID and actionable T-cell deficiencies with low TRECs,' economic evaluations would need to include the additional benefits and cost savings for identifying actionable T-cell lymphopenia at an early stage. As screening and diagnostic costs would remain unchanged, broadening the primary target disease would additionally result in a more favorable costeffectiveness ratio.
Economic evaluations should not only be used in decisionmodels of implementation of new disorders, but programs should update economic evaluations on a five-to-ten-year basis to reevaluate the program. International shared learning and publications on long-term outcomes might significantly improve these assumptions. Over time, technical advances might change screening and diagnostic test options, further improving outcomes after HSCT and new treatment modalities such as gene therapy might come into place. With changing demands and new developments, updates of existing economic evaluations are required for continuous optimization of NBS programs.

Worldwide overview of NBS for SCID
The first pilot studies for NBS for SCID were performed in the U.S.A. almost 15 years ago. The first state-wide SCID screening pilot study was initiated in Wisconsin in 2008 [68], with subsequent implementation of NBS for SCID in Massachusetts, Louisiana, and New York in 2009, and California, Texas, and Pennsylvania in 2010 [28]. NBS programs are a complex, multifaceted system, and introduction of a new condition can lead to disruption if all steps of the public health policy cycle are not carefully considered. Specifically for SCID, pilot studies were of great aid when introducing DNA analysis as a primary screening modality in NBS laboratories. In addition, these pilot studies were vital to the development of a strong evidence base to support decision-making regarding the addition of SCID to the NBS program of other countries.
In 2010 SCID was added to the RUSP which resulted in an acceleration in the number of states screening for SCID over the following years. By the end of 2018, NBS for SCID had been adopted by public health programs in all 50 states, the Navajo Nation, and Puerto Rico [52]. In 2011, the International Patient Organisation for Primary Immunodeficiencies (IPOPI) hosted the first event on NBS for SCID at the European Parliament. Pilot and proof-of-principle studies in Europe followed some years later in Sweden, United Kingdom, France, the Netherlands, and Spain [51,[69][70][71][72][73]. Nowadays, multiple nations around the world have instituted population-wide NBS for SCID, including but not limited to Israel, Iceland, New Zealand, Canada, Scandinavia, Taiwan, Germany, Switzerland, Czech Republic, and Japan. Others offer SCID screening in limited areas or have published preimplementation analyses and pilot studies [44,48,49,[74][75][76]. IPOPI has built an interactive tool to visualize the most up-todate status of NBS for SCID worldwide called the PID Life Index [14].

Harmonization of screening terminology for more efficient screening
Public health programs have a responsibility toward their stakeholders to continuously improve and optimize their NBS programs. Opportunities for improvement can be identified only if outcomes can be compared to unscreened populations or to other NBS programs. Comparison of screening outcomes has been hampered by use of disparate terminology and imprecise or variable case definitions for non-SCID conditions with T-cell lymphopenia. Standardization of terminology will promote international exchange of knowledge and optimize each phase of NBS and follow-up care. In 2022, the first recommendations were published on uniform case definitions in NBS for SCID reflecting literature and existing guidelines coupled with opinion of experts in public health screening and immunology [15]. By bringing together the previously unconnected public health screening community and clinical immunology community, these SCID NBS deliberations bridged the gaps in language and perspective between these disciplines. Implementing a new terminology on an international level might be complicated by the constraints of individual NBS programs. Different countries and even different regions vary greatly in various aspects of NBS programs. These differences can be explained by differing healthcare organizations, available resources, local politics, professional and patient groups, and policy makers [77,78]. There is little agreement among countries on which disorders should be included in NBS programs, but programs also differ in information provision procedures, time of sample collection, chosen screening tests and screening algorithms, and reporting abnormal test results and follow-up protocols [32,79]. Due to different resources and local regulations even treatment options might differ between countries. Harmonization of preanalytical, analytical, and postanalytical aspects of individual programs is not required, but all public health programs have a responsibility toward their stakeholders to continuously improve and optimize their NBS program. Harmonized registration of screening terminology and case definitions, is the first step that needs to be taken in order to recognize opportunities for improvement and to learn from other countries [15].

Conclusion
In the past decade, NBS for SCID based on the detection of TRECs has been introduced in many screening programs worldwide. SCID is the first IEI to be accepted for populationbased screening, and implementation has provided important clinical benefits for affected infants. Screening an entire population is a radical departure from previous paradigms in the field of immunology. Efficient screening strategies are costefficient and balance high sensitivity while preventing high numbers of referrals. NBS for SCID is accompanied by (actionable) secondary findings, but many NBS programs have optimized their screening strategy by adjusting algorithms or including second-tier tests. lymphopenia. Harmonization of terminology will promote international exchange of knowledge and optimize each phase of NBS and follow-up care.

Expert opinion
In the coming years, more and more NBS programs around the world are expected to include SCID in their screening program. However, implementation of the relatively expensive TREC assay may prove to be difficult for countries with limited resources. In some of these countries with high occurrence of consanguinity, the incidence of IEI is expected to be twenty times higher than in other countries. NBS for SCID would have a significant impact on the clinical outcomes of affected patients in these countries [80]. In order to move forward toward global NBS for SCID, it is essential to develop new, affordable screening tests that can screen for a broad range of neonatal conditions. Commercial efforts for innovative pricing of reagents and equipment can be of aid in these advances. Experts from various disciplines should lend their support by providing training and sharing their knowledge on a global scale [77]. NBS programs are of great importance to ensure early diagnosis of rare diseases, but if limited resources are available, it might be more feasible to focus on educational programs and public awareness. If NBS programs are not in place, clinicians should be made aware of early symptoms of IEI to enable early diagnosis and timely intervention [78]. Sharing experiences and strong cross-border collaborations between policy makers, clinical immunologists, pediatricians, and HSCT specialists, are vital for improved outcomes for SCID patients worldwide.
While some NBS programs are still awaiting governmental decisions about the inclusion of SCID, NBS programs that have already implemented SCID should continue to improve their current practice. Previous studies in the US showed a considerable number of SCID patients identified via NBS still developed infections prior to HSCT, suggesting that follow-up procedures could be further optimized [3]. To prevent significant delay in starting protective measures, time to obtain TREC results, to refer a newborn to a pediatric-immunologist and to obtain results of confirmatory testing should be reduced. It is recommended to harmonize best practices for isolation and antimicrobial prophylaxis to minimize infection exposure before HSCT.
In the next 5 years, many NBS programs may have implemented a multiplex PCR, measuring TRECs simultaneously with kappa deleting recombination excision circles (KRECs) and SMN1, a marker for SMA [67]. Measuring KRECs with NBS could potentially identify severe B-cell deficiencies such as XLA at birth [79,81]. It has yet to be determined whether an early diagnosis of XLA will lead to better health outcomes or improved quality of life. In addition, the sensitivity of KREC screening would have to be increased by including second-tier testing to prevent many falsepositive referrals. A multiplex TREC/KREC assay could additionally aid in the diagnostic process by distinguishing B-/B + phenotypes in SCID patients. With TREC-only screening, delayed-onset or atypical SCID patients, such as T-B-SCID patients with hypomorphic mutations in cellular metabolism or DNA repair might be missed [82]. B-cells are more vulnerable to genomic stress caused by the increase of toxic metabolites in ADA-SCID in comparison to T-cells potentially leading to absent KRECs. Tandem mass spectrometry measuring SCID screening might also be adenosine and deoxyadenosine for ADA deficient patients or purine nucleosides and 2′-deoxy-nucleosides for PNP deficient patients might be an additional technique to identify these patients. Previous studies have shown that screening with tandem mass spectrometry was able to identify these infants at low cost [54,55,83,84]. The question remains whether PNP is a suitable candidate for NBS given that T-cell counts are not absent at birth, but rather decline progressively over time.
Expansion of NBS with new disorders is driven by development of new test modalities and treatment options. After expansion of the above-mentioned tandem mass spectrometry panels, improved efficiency and throughput time of epigenetic immune cell counting could offer early detection of several IEI with quantitative defects of immune cell populations shortly after birth. SCID was the first IEI in population-based screening and at the same time the TREC assay became the first highthroughput DNA-based test in NBS laboratories. In addition to SCID, there are many other IEI that could benefit from early diagnosis and intervention if a suitable NBS test was available. With the Wilson and Jungner screening criteria in mind, several IEI would qualify as serious conditions that cause an important health problem and would benefit from early detection and treatment by preventing severe infections, early onset severe immune dysregulation, and auto-immunity [13,85].
In the coming years, NBS for IEI will enter the genomic era. First-tier WGS-based screening in newborns has its challenges to overcome, but more and more countries will include NGS with IEI panels in their NBS programs as a second tier test in the next years. First-tier NGS in NBS and the accompanied ethical, social, legal implications (ELSI) and costs will have to be further explored in multistakeholder studies. In the genomic era, genome-wide associations studies may have identified an exceeding number of associations between variants and phenotypes. Pathogenicity prediction programs will demonstrate improved accuracy and reference databases will be more complete. Neonatal screening might no longer rely on dried blood as DNA can be collected through less invasive methods, such as saliva or oral mucosa. In this era, nonactionable diseases might be included in the NBS program to avoid long diagnostic odysseys. In addition, NBS for early-onset diseases might have been complemented with conditions presenting in adulthood conflicting with the 'child's right to an open future.' Even risk scores of potentially developing a certain disease at some stage in life might be reported early in life. While the future of NBS may be uncertain, one thing is clear: population-based screening programs have exciting times ahead thanks to the countless technological advances that lie ahead.