Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders comprising more than 240 different pathologies, with an approximate incidence of 1:500 live births. Congenital defects of the immune system, such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinaemia (XLA) are less frequent, although they still have a considerable incidence of 1:70000 to 1:100000 live births.1,2 The vast majority of neonates with severe T- and/or B-cell lymphopaenias, including those suffering from severe PIDs, have no family history of PID and are asymptomatic in the first weeks of life.3 Due to these features, delayed diagnosis is frequent, and it is associated with considerable morbidity and mortality due to the development of severe clinical manifestations at a later point.2–4 There are highly efficacious treatment options for the most severe forms of PID, for instance, immune globulin replacement therapy for the agammaglobulinaemias; and curative treatments for SCIDs, such as haematopoietic stem cell transplantation or gene therapy.5,6 It has been demonstrated that early diagnosis followed by implementation of preventive and therapeutic measures reduces the rate of sequelae, improves the quality of life of patients, and dramatically improves survival outcomes.7,8 Likewise, early identification of patients with SCID helps prevent severe iatrogenic damage, for instance by avoiding administration of live vaccines (such as the rotavirus vaccine) currently recommended to start at 2 months of age, when most patients have yet to develop characteristic symptoms.9

The methodology needed to detect PID routinely in the neonatal period has been developed in recent years. In United States, real-time PCR has been used to identify patients with SCID by quantification of T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples.10–12 Recently, pilot studies have been conducted in Sweden and Germany to evaluate a method that allows detection of T-cell lymphopaenias (through low levels of TRECs), that are typically associated with SCID, and B-cell lymphopaenias (through low levels of kappa-deleting recombination excision circles [KRECs]) associated with other severe PIDs such as XLA or ataxia telangiectasia (AT).13–16 TRECs and KRECs are episomal DNA fragments that result from gene rearrangements that occur during T- and B-lymphocyte maturation. The excision circles do not replicate during mitosis and thus exhibit a dilution pattern that allows the quantitative estimation of cell replication.12,16 Thus, patients suffering from severe PIDs who have T- and/or B-cell lymphopaenias have very low levels of TRECs and KRECs, regardless of the molecular aetiology of the PID, which is very useful considering the genetic heterogeneity of these disorders.10,13,16

In Spain, neonates are not routinely screened for potentially lethal diseases of the immune system, despite evidence that these pathologies meet the screening criteria adapted from Wilson and Jungner, which determine the appropriateness of including specific diseases in population screenings: high morbidity and mortality, an initial latent stage, availability of an accessible and relatively non-invasive screening method, evidence of benefits of early diagnosis, availability of curative or effective treatment, and finally, a favourable cost-benefit analysis.17–19

Due to a lack of previous studies and to shortfalls in the data of national registries, we do not know the real current prevalence of severe PIDs in Spain. The results of neonatal screening for PIDs in California changed what was known about the prevalence of SCID, showing a rate of 1:70000 live births compared to previous estimates ranging from 1:100000 to 1:150000 live births.20

This paper presents the initial data of a pilot study with the purpose of: (a) finding out the magnitude of an unidentified problem and (b) providing data on the feasibility and performance of a novel methodology using samples that had been obtained previously for the established neonatal screening programme.

In a second phase of the study with a larger sample we hope to: (a) obtain reliable information on the epidemiology of T- and/or B-cell lymphopaenia in our setting; (b) learn about the aetiology of T- and/or B-cell lymphopaenia, and, lastly (c) identify neonates with lymphopaenias who could benefit from early treatment, thus improving the prognosis of patients suffering from a PID.

Patients with severe PIDs benefit from early diagnosis, as it enables prompt implementation of preventive, supportive, and therapeutic measures that improve their overall prognosis and, in the case of patients with SCID, improve survival rates through hematopoietic stem cell transplantation.5–8

Previous studies have demonstrated the reliability of the methodology based on TRECs determination in heel blood samples for identifying patients suffering from severe T-cell lymphopaenia.13,20 Considering this evidence, European experts in PID have expressed their unambiguous support for the introduction of this screening test in Europe.21 Recently, Modell et al. presented cost-benefit data that greatly favoured the implementation of neonatal screening for SCID, and proposed a model to estimate its socioeconomic impact for different populations.18 Their calculations, based on screening data from the United States, showed that the total healthcare cost for a patient with delayed diagnosis amounted to $6000000; while the cost of mass screening and the treatment and follow-up of one patient diagnosed following the screening amounted to $1385000.18 It is worth noting that the cost per neonate of the TRECs and KRECs screening in our study was €1.46, and therefore well below the cost of screening in the United States ($4.25/neonate) and the United Kingdom (£8.30/neonate), where the screening only includes the TRECs assay.

The potential of KRECs to be a marker for disorders of B-lymphocyte development in the field of routine neonatal screening has been reported in the literature.13,22 By determining TRECs and KRECs simultaneously in a single assay, its diagnostic capacity increases. Multiplex PCR (TRECs/KRECs/ACTB assay) can identify classic congenital T-lymphocyte defects (SCID), but also other immune disorders in asymptomatic newborns, such as agammaglobulinaemias, combined immunodeficiencies, and AT.14,21 The assay can also identify patients with an atypical SCID phenotype that could go undetected in assays that only determine TREC levels.23

In our pilot study, we analysed 1088 samples from 1068 neonates born in southern Spain with the aim of knowing the levels of TRECs and KRECs in this population, identifying suitable cut-off points, and assessing the feasibility of the TRECs/KRECs/ACTB assay in our setting.

Recent studies in the United Kingdom, France, and Israel, most of them retrospective, suggest that it would be beneficial to add screening for SCID to the routine neonatal screenings.22,24,25 Due to its prospective design, our study is one of the first of its kind not only in Spain, but also in Europe.

The TRECs and KRECs values observed were comparable to those previously described in other populations based on the same methodology.13 At first we used conservative cut-off values of TRECs<15/μL and KRECs<10/μL. With these cut-offs, 0.75% of the tested samples had abnormal results in our population. Other groups have reported figures between 0.08% and 4.1%, depending on the cut-off points used.13,20,21 Population-based studies with a high number of abnormal samples would be unfeasible, because of our healthcare area (with 25000 births/year) such a rate would entail having 188 samples retested every year. There are 3 possible reasons why the retest rate is this high: (a) low quality of the DBS samples taken before the analysis; (b) the learning curve for the methodology; and finally (c) conservative cut-off points. When the abnormal samples were retested, all but one gave results above the initial cut-off points (0.09%). This recall rate is considered adequate for this type of study.19

When we considered more restrictive cut-off points (TRECs<8/μL and KRECs<4/μL), we observed that all samples tested normal (not pathological). Recent data published by Gaspar et al. in the United Kingdom, and data by Borte et al. (personal communication) are similar and suggest that a specific cutoff point needs to be determined for each laboratory and population.20,26

The ultimate goal is to reduce the retest rate. To achieve this, it is important that an effort is made to obtain quality samples and to improve their quality, and that a suitable cut-off point for the Spanish population is determined. This requires ongoing communication with the associated healthcare centres. Reducing the retest rate would prevent unnecessary costs and worry in families, and in particular cases, it would preclude the need to collect a new blood sample from the newborn (recall).

Preterm neonates may get false positive results more frequently with this assay, so they need to be managed differently, as specified in Fig. 1. Our pilot study found no significant differences between the two birth weight subgroups (<2500g vs ≥2500g) and the two gestational age subgroups (<37 weeks vs ≥37 weeks), although we need to consider that our population did not include samples from extremely preterm neonates (birth weight <1000g or gestational age <28 weeks). For now, given the controversy in the literature, it seems prudent to maintain a conservative approach in neonates born preterm or requiring intensive care who have received positive results.13,22,23

Out of the four controls provided by the CDC, we correctly identified two normal samples and two abnormal samples compatible with a T−B− SCID phenotype, one of them with low ACTB copy numbers, which confirmed the diagnostic capacity of the assay.

Samples of patients previously diagnosed with XLA and AT were also analysed. Abnormal results were found for both pathologies. Patients with XLA were identified through very low copy numbers of KRECs, and both patients with AT should TRECs and KRECs values be below the established cut-off points, as had been observed in previous studies.13,15 Since these samples were not collected in the neonatal period, their value to validate the cut-off point for these pathologies is limited. Nevertheless, the fact that they gave abnormal results in the screening is worth noting, as the patients with AT (ages 1 and 17 years) had significant lymphopaenia and required prophylactic treatment against Pneumocystis jirovecii and immune globulin replacement therapy.

Last of all, we analysed a sample obtained from a patient recently diagnosed with T−B+ SCID. It is noteworthy that all patients presented clearly abnormal levels of TRECs and/or KRECs (0–1copy/μL) well below the cut-off points.

This paper is the first to present results of a prospective pilot study that used an innovative methodology on dried blood samples from Spanish newborns to identify those with severe T- and B-cell lymphopaenias. The study provides valuable information on the feasibility and diagnostic capacity of this methodology. The prospective and continuous recruitment of neonatal samples will allow us to increase the validity of the obtained data, which will help implement neonatal screening for severe PIDs at the regional and eventually national levels. The cooperation of different groups of providers in the healthcare system (nurses, neonatologists, primary care paediatricians, paediatric specialists, immunologists, and experts in metabolic disorders) is essential for its implementation to be successful. The ultimate objective is to be able to diagnose asymptomatic newborns with severe PIDs in the first weeks of life. This will allow prompt initiation of preventive and supportive measures, and even of curative treatment, improving the quality of life of these patients and their families.

1. Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III (PI13/01104).

2. Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla (FISEVI, “Scott Family Funding”).

3. Río Hortega grants for hiring research interns. Instituto de Salud Carlos III.

4. German Federal Ministry of Education and Research (BMBF 1315883).

P. Olbrich and B. de Felipe contributed equally to this work.

The authors have no conflicts of interest to declare.