ORIGINAL RESEARCH published: 22 October 2019 doi: 10.3389/fimmu.2019.02406 First Universal Newborn Screening Program for Severe Combined Immunodeficiency in Europe. Two-Years’ Experience in Catalonia (Spain) Ana Argudo-Ramírez 1† , Andrea Martín-Nalda 2† , Jose L. Marín-Soria 1 , Rosa M. López-Galera 1 , Sonia Pajares-García 1 , Jose M. González de Aledo-Castillo 1 , Edited by: Mónica Martínez-Gallo 3 , Marina García-Prat 2 , Roger Colobran 3,4 , Jacques G. Riviere 2 , Antonio Condino-Neto, University of São Paulo, Brazil Yania Quintero 1 , Tatiana Collado 1 , Judit García-Villoria 1 , Antonia Ribes 1 and Pere Soler-Palacín 2* Reviewed by: Raz Somech, 1 Newborn Screening Laboratory, Inborn Errors of Metabolism Division, Biochemistry and Molecular Genetics Department, Sheba Medical Center, Israel Hospital Clínic, Barcelona, Spain, 2 Pediatric Infectious Diseases and Immunodeficiencies Unit, Jeffrey Modell Diagnostic and Lisa Renee Forbes, Research Center for Primary Immunodeficiencies, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Baylor College of Medicine, Barcelona, Spain, 3 Immunology Division, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, United States Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Department of Clinical and Mirjam van der Burg, Molecular Genetics, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Hospital Universitari Vall Leiden University Medical d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain Center, Netherlands *Correspondence: Pere Soler-Palacín Severe combined immunodeficiency (SCID), the most severe form of T-cell psoler@vhebron.net immunodeficiency, can be screened at birth by quantifying T-cell receptor excision † These authors have contributed circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition equally to this work speeds up the establishment of appropriate treatment and increases the patient’s life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Specialty section: Spanish and European region to universally include this testing. The results obtained in This article was submitted to Primary Immunodeficiencies, the first 2 years of experience are evaluated here. All babies born between January 2017 a section of the journal and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) Frontiers in Immunology was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In Received: 27 May 2019 Accepted: 25 September 2019 2018, the retest cutoff in the detection algorithm was updated based on the experience Published: 22 October 2019 gained in the first year, and changed from 34 to 24 copies/µL. This decreased the retest Citation: rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate Argudo-Ramírez A, Martín-Nalda A, of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK Marín-Soria JL, López-Galera RM, Pajares-García S, González de populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αβ Aledo-Castillo JM, Martínez-Gallo M, and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation García-Prat M, Colobran R, Riviere JG, Quintero Y, Collado T, were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 García-Villoria J, Ribes A and tested positive, 15 of which were male. During the study period, one patient was Soler-Palacín P (2019) First Universal diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients Newborn Screening Program for Severe Combined Immunodeficiency had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 in Europe. Two-Years’ Experience in newborns (43% of positive detections). Nine patients were considered false-positive Catalonia (Spain). Front. Immunol. 10:2406. cases because of an initially normal lymphocyte count with normalization of TRECs doi: 10.3389/fimmu.2019.02406 between 3 and 6 months of life, four infants had transient lymphopenia due to an initially Frontiers in Immunology | www.frontiersin.org 1 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia. Keywords: newborn screening, severe combined immunodeficiency, T-cell receptor excision circles, T-cell receptor, T-lymphocytes, stem cell transplantation INTRODUCTION forms of B cell deficiency, such as X-linked agammaglobulinemia (XLA) (14). Newborn screening (NBS), initially implemented in the The implementation of screening through TREC assays United States in 1961 (1), has been available in most developed provides the earliest possible identification and allows for countries for decades, originally for the detection and early prompt, successful transplantation before infants experience treatment of phenylketonuria and now for other endocrine and severe infection, organ damage, and, ultimately, death (15). metabolic diseases, mainly using tandem mass spectrometry. The cost-effectiveness of including SCID in NBS programs DNA-based testing for other diseases has been only recently has been demonstrated in the United States (16, 17) and included in the NBS programs of some countries. Since Europe (6, 10, 18, 19). its initial implementation in Wisconsin in 2008, NBS for In Catalonia, the NBS program started with phenylketonuria severe combined immunodeficiency (SCID) using a T-cell detection in 1969 and congenital hypothyroidism in 1982. receptor excision circle (TREC) assay has been established Today, around 67,000 newborns per year are screened for worldwide, including in most of the United States (2), Taiwan 24 diseases (phenylketonuria, congenital hypothyroidism, cystic (3), Israel (4), New Zealand, some Canadian regions (2), fibrosis, sickle cell disease, aminoacidopathies, organic acidurias, Norway (5), and Catalonia (Spain). Previously published mitochondrial beta-oxidation disorders, and, lately, SCID). SCID results showed an incidence of SCID of around 1 in 56,000 was included in the Catalonian NBS program in January 2017, newborns (5, 6) and a high survival rate (92%) after appropriate and data from the first 24 months following its implementation treatment (2). are presented here. SCID, the most severe form of T-cell primary immunodeficiency (PID), includes a group of inherited defects characterized by severe T-cell lymphopenia (TCL). Patients with SCID require prompt clinical intervention to prevent severe MATERIALS AND METHODS life-threatening infections, and several studies have reported Population and Data Collection significantly improved survival in babies diagnosed at birth All consecutive DBS samples received as part of the universal (7, 8). Curative treatment is based on hematopoietic stem cell NBS program in Catalonia between January 2017 and December transplantation (HSCT) or gene therapy when available (8). 2018 were analyzed (n = 130,903). Samples with the following SCID can be screened at birth in a cost-effective way on a large characteristics were excluded: collection time before 44 h or after scale through quantification of T-cell receptor excision circles 7 days of life, transfusions, poor DNA amplification, and poor (TRECs) in Guthrie card dried blood spot (DBS) samples (9, 10). quality or blood amount. Ultimately, this study was performed in SCID newborn screening can also identify other clinically 129,614 newborns. relevant forms of TCL, which have an overall incidence of 1 Demographics (birth date, date of sample collection, parents’ in 7,300 newborns (2). These include specific syndromes such origin, newborn sex, gestational age, and birth weight) were as 22q11 deletion (DiGeorge) syndrome, Down syndrome, and electronically collected. Extremely preterm newborns were CHARGE syndrome among others (11, 12). defined as those with a gestational age <32 weeks, preterm TRECs are circular DNA molecules formed by T-cell receptor newborns ≥32 and <37 weeks, and term newborns ≥37 weeks. gene rearrangement during the normal process of T-cell Low birth weight in term babies was defined as <2,500 g and differentiation in the thymus. TRECs do not undergo further normal birth weight as ≥2,500 g. replication in dividing cells. Hence, they are stable circular DNA From 1 January to 30 June 2017, newborns (n = 33,040) fragments, which are a useful marker of recently formed T-cells, underwent SCID screening as part of a 6-month prospective a cell population that is extremely low in newborns with SCID. implementation pilot study that validated our approach. TREC copy number can be determined using a quantitative However, in January 2018, we decided to update the decision PCR-based method with time-resolved fluorescence resonance algorithm (Figure 1), lowering the retest cutoff from 34 to energy transfer (TR-FRET)-based detection in eluted DNA 24 copies/µL. The results from newborns screened in 2018 from routinely collected DBS (9). Kappa-deleting recombination were then evaluated (n = 64,092; 63,393 after applying excision circles (KRECs), the circular by-product of B cell exclusion criteria). immunoglobulin kappa gene rearrangement, have been proposed The study was approved by the Government of Catalonia for a combined TREC-KREC screening approach (13). This (Departament de Salut, Generalitat de Catalunya) for its NBS could also enable the early detection of patients with severe program. Specific informed consent was obtained from all Frontiers in Immunology | www.frontiersin.org 2 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia FIGURE 1 | SCID NBS detection decision algorit hm. *The retest cutoff was changed from 34 to 24 copies/µL in 2018. **If beta-actin gene <50 copies/µL a second sample was requested because the sample was considered of unsatisfactory quality. TRECs, T-cell receptor excision circles. individual participants included in the study whose genetic with blanks and three DBS calibrators was run in triplicate on evaluation was needed. each plate. A low-TREC control, a no-TREC control, a high- TREC control, and a blank paper disk (with no blood content) were used as quality controls in each plate. Interpretation of the Sample Testing assay results uses fluorescence counts measured at 615, 665, and Quantification of TRECs in DBS (1.5-mm diameter spot) 780 nm. Corrected fluorescence counts, TREC responses, and was performed according to the commercial Enlite Neonatal beta-actin responses for all reactions were calculated from the TREC kit instructions (PerkinElmer, Turku, Finland). The raw fluorescence counts. Calibrator responses were fitted against EnLite TREC kit is a combination of PCR-based nucleic acid the ArcSinh transformed concentrations (copies/µL) using amplification and time-resolved fluorescence resonance energy unweighted linear regression. The Enlite software generated a transfer (TR-FRET)-based detection. The assay detects two calibration curve and the sample and DBS control concentrations targets simultaneously: TRECs and beta-actin, which is used (copies/µL) in each run. as the internal control to monitor specimen amplification in each test. DBS punches were directly inserted in 96-well PCR plates Definition and Interpretation of Results using a Wallac DBS puncher (PerkinElmer). DNA was eluted in Before implementing the protocol, the method was verified the first step, and the second step involved PCR amplification (repeatability, reproducibility, limit of detection, limit of of TRECs and beta-actin as well as hybridization with target quantification, sensitivity, and specificity) and these parameters sequence-specific probes. The PCR plate was then read in a were successfully compared with those stated in the kit Victor EnLite fluorometer (PerkinElmer). A full calibration curve insert (Supplementary Material S1). In addition, available NBS Frontiers in Immunology | www.frontiersin.org 3 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia FIGURE 2 | Immunological and genetic protocol in positive cases. 1 Flow cytometry protocols are shown in Supplementary Material S5; 2 Lymphopenia and SCID criteria are shown in Supplementary Material S2; 3 AFP values are not reliable before 12 months of age; therefore, AFP was only studied in patients older than one year lacking an alternative diagnosis; 4 The list of 323 PID genes is shown in Supplementary Material S3. AFP, alpha-fetoprotein; ATM, ataxia telangiectasia; B, B-cells; CMV, cytomegalovirus; Ig, immunoglobulin; NK, natural killer cells; PID, Primary immunodeficiency; SCID, severe combined immunodeficiency; T, T-cells; TCR, T-cell receptor; TRECs, T-cell receptor excision circles. samples from children with a known SCID diagnosis in Catalonia considered as positive detection (retest cutoff changed from 34 to in the last 5 years were analyzed as positive controls (n = 6; 24 copies/µL in 2018). These positive detections were notified to median, range TREC copies/µL: 2, 2–4), as well as five other the SCID Clinical Reference Unit (SCID-CRU) to initiate clinical positive samples from the SCID Newborn Screening Quality and immunological evaluation. Assurance Program-Proficiency Testing Program provided by The retest after the first sample rate (retest rate), requested the CDC (Centers for Disease Control and Prevention, second sample rate, and SCID-positive detection rate (positive Atlanta, USA). detections) were calculated. Based on these results, the algorithm After reviewing the decision algorithms from other NBS was reevaluated. Validation of the results was carried out using programs with previous experience of this disease, we decided to Specimen Gate (Perkin Elmer) and Nadons (Limit4, Barcelona, start the pilot study with the algorithm used by Audrain et al. (6), Spain) software. which had a threshold of >34 copies/µL (Figure 1). Samples whose TREC value was ≤34 copies/µL were retested Initial Clinical and Immunological in duplicate. If two of the three values were ≤20 copies/µL, a Assessment at SCID-CRU second sample was requested. Samples with TRECs ≤5 copies/µL Within the first 7 days after detection, all positive cases were (preterm infants) or ≤10 copies/µL (term newborns) in the first referred to SCID-CRU, where clinical and immunological sample (both with beta-actin gene ≥50 copies/µL), as well as assessment was performed per protocol (Figure 2). analyses with TRECs ≤20 copies/µL in the second sample, were Complete family and medical histories were recorded, and Frontiers in Immunology | www.frontiersin.org 4 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia a meticulous physical examination was carried out. In addition, TABLE 1 | Demographic data and TREC values in the study population. psychological support was offered to parents, starting with this Sample size n = 129,614a TRECs, p-valuec first visit. copies/µL, Several tests were performed: T, B, and NK cell median (IQR) immunophenotyping, expression of CD45RA/RO, TCR αβ, and γδ, HLA-DR+ expression on T lymphocytes, in vitro All newborns 104 (68-162) – lymphocyte proliferation, and immunoglobulins including IgE. Sex SCID criteria (Supplementary Material S2) were defined as Male 51.5% (66, 751) 98 (64–152) <0.05 reported by Kwan et al. (2). In cases of non-SCID lymphopenia, Female 48.5% (62, 863) 111 (73–173) a consultation was scheduled with the geneticist for clinical Sample collection, hours, 50 (49–60) – – evaluation and array-based comparative genomic hybridization median (IQR) (CGH-array) studies. If these results were normal, a genetic Gestational age, weeks, 39 (38–40) panel covering most PID was performed. We used a custom- median (IQR) designed next-generation sequencing (NGS)-based panel that Extremely preterm, <32 0.6% (830) 70 (43–127) <0.05 weeks, % (n) targets 323 genes (Supplementary Material S3), including Preterm, 32-36 weeks, % 5.9% (7,561) 96 (62–155) most of the known PID-causing genes according to the (n) 2017 IUIS (International Union of Immunological Societies) Term, ≥37 weeks, % (n) 93.5% (121, 223) 105 (68–162) classification, and other genes recently described as causing Birth weight in term 3300 (3,010–3,590) PID (20). This panel has been successfully used in our newbornsb , grams, laboratory over the last few years for genetic diagnosis of median (IQR): PID (21). Low birth weight, % (n) 3.4% (4,385) 94 (61–145) <0.05 Normal weight,% (n) 96.6% (114, 777) 105 (69–163) Statistical Analysis a Sample size after applying exclusion criteria; b Term newborns (n = 121, 223). c Mann- Statistical analyses were performed using SPSS software version Whitney U or Kruskal-Wallis test were used for group comparisons (p-value calculated at 23.0 (SPSS Inc., Chicago, Illinois). Given the non-parametric a 5% significance level). IQR, interquartile range. distribution of the data, the Mann–Whitney U or Kruskal– Wallis test were used for group comparison analyses (25 and 75th percentiles in brackets). Two-tailed statistical analysis Cutoff Values and Rates was performed, and differences were considered statistically The overall results from 2017 to 2018 were retest rate 2.4%, significant at p < 0.05. requested second sample rate 0.23%, and positive detection rate 0.02% (Figure 3). With the application of the initial TREC retest cutoff of 34 RESULTS copies/µL (3rd percentile in the 2017 population), results from Demographics the 66,214 samples analyzed during 2017 were as follows: retest Of the 129,614 newborns screened after applying the exclusion rate 3.34% (n = 2212), requested second sample rate 0.21% (n = criteria (initial n = 130,903), 51.5% were male and 48.5% were 138), and positive detection rate 0.02% (n = 16). female. The median age at sample collection was 50 h of life After reducing the retest cutoff to 24 copies/µL (1st (interquartile range [IQR], 49–60). Median gestational age was percentile), the retest rate was 1.4% (n = 898). The detection 39 weeks (38–40): 0.6% were extremely preterm newborns (n = cutoff of 20 copies/µL remained the same (0.7th percentile), and 830), 5.9% preterm (n = 7,561), and 93.5% term newborns (n the requested second sample and positive detection rates were = 121,223). Median birth weight in term newborns was 3,300 g similar to those of 2017. (3,010–3,590) (Table 1). Median TREC value in the study population was 104 Positive Detections copies/µL (IQR: 68–162), with 20 copies/µL being the 0.7th There were 30 positive detections in the 129,614 screened percentile. Median TREC values showed statistically significant newborns: 40% (n = 12) were detected in the first sample differences in relation to the patients’ sex, gestational age, and and 60% (n = 18) in the second, with 50% (n = 15) of birth weight (only assessed in term babies). Median TREC values detections occurring in males. There was no history of maternal were 98 (64–152) and 111 (73–173) in males and females, immunosuppression in any of the positive cases. respectively (p < 0.05). In relation to gestational age, the Newborns with positive detection were referred to the median TREC value demonstrated a progressive increase: 70 (43– SCID-CRU per protocol. The various diagnoses in the 30 127), 96 (62–155), and 105 (68–162) copies/µL in extremely patients were as follows (Figure 4): SCID (1/30), partial 22q11 preterm, preterm, and term newborns, respectively (p < 0.05) DiGeorge syndrome (5/30), idiopathic lymphopenia (3/30), (Supplementary Material S4). Regarding birth weight in term chylothorax (2/30), prematurity (2/30), and Down syndrome newborns, median TREC was 94 (61–145) and 105 (69–163) (1/30). Laboratory and clinical data of non-SCID TCL patients copies/µL in low and normal birth weight babies, respectively are described in Table 2. Nine patients were considered to (p < .05) (Table 1). have false-positive results (i.e., initially low TRECs and normal Frontiers in Immunology | www.frontiersin.org 5 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia FIGURE 3 | Results of SCID NBS in Catalonia over the study period (2017–2018). 1 2018 cutoff (2017 cutoff = 34 copies/µL); 2 2017+2018-Retest rate 2017-Retest rate = 3.34% (cutoff = 34 copies/µL); 2018-Retest rate = 1.4% (cutoff = 24 copies/µL); 3 Three patients are currently under study. NB, newborns; SCID, severe combined immunodeficiency; SCID-CRU, SCID Clinical Reference Unit. lymphocyte count, with normalization of TRECs between 3 and a 2-year experience and more than 130,000 newborns screened, 6 months of life), four patients had transient lymphopenia (i.e., one SCID patient and 13 other conditions were identified. In initially low TRECs and low lymphocyte count, with recovery addition, we were able to consolidate our detection strategy in the following months), and three patients are currently and the characterization, diagnosis, and follow up protocol for under study (SCID excluded, but requiring additional TREC positive cases. testing at 3 and 6 months of life). The incidence of clinically As a pioneer initiative in Europe, we encourage the definition significant non-SCID TCL was 1 case in 10,069 newborns (43% of a single technical approach for SCID newborn screening to of positive detections). facilitate future comparative studies between different countries. Thus, the incidence of SCID was 1 in 130,903 newborns. With this in mind, we decided to implement the commercially The SCID patient was a Caucasian male of 35 weeks’ gestation available EnLite Neonatal TREC kit (Perkin Elmer, Turku, with 0/0 TREC copies/µL in DBS. Lymphocyte count was 0.4 Finland), which has been validated elsewhere and has shown × 109 /L with a T-B- NK+ phenotype and an absent in vitro comparable median TREC values between different studies (4, 6, proliferative response to mitogens. Whole exome sequencing was 22, 23). This kit allows for DNA elution and gene amplification performed, but no causative mutations were found. However, a in a single process, as well as standardization and reproducibility variant of uncertain significance was identified in the LIG1gene. according to ISO 15189 standards. With the aim of minimizing The patient underwent HSCT at the age of 2 months of life using the previously described inter-lot variability (6), we established reduced intensity conditioning, with a good clinical outcome and a retest cutoff higher than the request second sample cutoff, immunological reconstitution. which provided a range between the retest and detection cutoffs. The initial retest cutoff was 34 copies/µL (3rd percentile), which was changed to 24 copies/µL (1st percentile) in 2018, DISCUSSION whereas the request second sample cutoff was maintained at 20 copies/µL. This study reports the Catalonian NBS experience for SCID The initial retest cutoff of 34 copies/µL (3rd percentile) detection based on TREC quantification, and enables detection led to a retest rate of 3.34%, which was considered too high of non-SCID lymphopenic conditions in addition to SCID. After in comparison with data from other authors (24). Hence, we Frontiers in Immunology | www.frontiersin.org 6 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia then evaluated the first percentile from our population (24 copies/µL were normal: 1,455 (98.4%) after duplicate repetition copies/µL) as a new retest cutoff. We found that 1,477 of and 22 (1.6%) after a second DBS analysis. Only one baby 1,478 babies with initial TREC values between 34 and 25 within this range was referred for study in the SCID-CRU and the diagnosis was not a case of SCID, the objective of this screening. Following this analysis, we decided that it was reasonable to switch to 24 copies/µL as the TREC repeat cutoff, implemented since 2018. That change reduced the SCID Under study (1/30) repetition rate to 1.4%, now comparable to the rate described (3/30) Partial 22q11.2 by others (24) and led to a reduction in the screening cost DiGeorge syndrome per patient. (5/30) Our internal algorithm showed a second sample request rate and positive detection rate of 0.2 and 0.02%, respectively, in keeping with the rates in previous studies, as summarized in the review by van der Spek et al. (24). In addition, the False positives1 (9/30) Transient percentiles of our population corresponding to each cutoff are lymphopenia2 (4/30) comparable to those of other diseases included in our program, such as congenital hypothyroidism, phenylketonuria, and other metabolic diseases. Of note, the method was unable to adequately quantify beta-actin copies in some of the external quality control Idiopathic samples from the CDC program, a shortcoming that may lymphopenia Down syndrome (3/30) be related to PCR inhibition, as was suggested by the CDC (1/30) Prematurity Chylothorax itself. Nonetheless, this limitation did not affect the results of (2/30) (2/30) SCID screening. Several studies have used combined TREC and KREC FIGURE 4 | Final diagnoses in patients testing positive in Catalonia (2017–2018). 1 False-positives due to initially normal lymphocyte count with screening assays to simultaneously detect T- and B-cell defects normalization of TRECs between 3 and 6 months of life; 2 Transient (18, 25), including a pioneer study performed in Spain by de lymphopenia due to initially low lymphocyte count with recovery in the Felipe et al. (26), but we decided to focus on classical SCID following months. Final diagnosis of the 13 non-SCID lymphopenic patients patients who completely fulfill the NBS criteria, in accordance are now boxed. SCID, severe combined immunodeficiency. with the experience in the United States (2, 5). Although TABLE 2 | Clinical and laboratory data in non-SCID TCL patients. Patient Gestational Sex Prenatal TREC levels Lymphocyte Lymphocyte Lympho- Clinical features at Diagnosis age diagnosis (copies/µL) count, (x109 /L)* subsets:T cells/B proliferationassay birth cells/NK cells (x109 /L)** 1 40 Female No 17 4.2 2.1/19.9/1.1 Normal None 22q11.2 DS 2 40 Female No 5 5.4 1.6/1.1/2.3 Normal None 22q11.2 DS 3 34 Female Yes 5 2.6 1.1/0.6/0.7 Normal Congenital heart disease 22q11.2 DS 4 39 Female Yes 17 2 1.9/1.4/2 Normal Congenital heart disease 22q11.2 DS and hypocalcemia due to hypoparathyroidism 5 41 Female No 17 1.7 1.2/0.04/0.3 Normal None 22q11.2 duplication 6 40 Male No 14 3.7 2.2/0.6/0.6 Normal None Idiopathic lymphopenia 7 40 Male No 7 3.3 1.7/0.6/0.7 Normal None Idiopathic lymphopenia 8 35.2 Male No 6 1.7 2/0.2/0.2 Normal None Idiopathic lymphopenia 9 34 Female Yes 6 7.8 NA Normal Congenital Chylothorax Chylothorax 10 34 Female Yes 4 7.4 NA Normal Hydrops fetalis Chylothorax 11 32 Male No 7 2.7 2.9/0.8/0.2 Normal None Prematurity 12 29 Male No 18 4.4 2.9/1/0.2 Normal None Prematurity 13 31 Female No 15 3 NA Normal Esophageal atresia Down syndrome * Reference value: 3.4–7.6; ** Reference values: T-cells: 1.8–5.9; B-cells: 0.6–1.9; NK-cells: 0.1–1.3. CGH, comparative genomic hybridization; DS, deletion syndrome; NA, not available; NBS, newborn screening; SCID, severe combined immunodeficiency; TREC, T-cell receptor excision circle. Frontiers in Immunology | www.frontiersin.org 7 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia inclusion of KREC values to the TREC assay would enable the psychologist provided support starting from the first contact with detection of patients with hypomorphic mutations leading to a the family in a joint visit with the pediatric immunologist. The leaky SCID phenotype and delayed-onset adenosine deaminase psychologist’s task was to guarantee that parents were able to (ADA) deficiency (14, 25, 27), it might also result in an increase understand and assimilate the information about the possible in false-positive testing and higher recall and retest rates. diagnosis and the process to follow in the event that it was Our data support the previously reported influence of confirmed. The family was offered a space for containment and gestational age and sex on TREC values (27–30). Thus, in the support for the impact of the diagnosis, and strategies were case of low values in preterm babies or low-birth-weight term activated to control anxiety. The psychologist maintained contact newborns (i.e., TREC values above 5 copies/µL in preterm babies to ensure adherence to the treatments prescribed and compliance and 10 copies/µL in term newborns), cautious evaluation and a with the control visits and tests. Support was intensified in request for a second sample are needed at term and when birth the most complicated moments, such as hospital admission weight reaches >2,500 g, respectively. To our knowledge, there is and isolation, and parents were accompanied in all phases no explanation for the significantly higher TREC values seen in related to transplantation. Several cases of parental anxiety were female newborns; however, these differences have been described detected and appropriately managed. In the yearly evaluation, previously in a cohort study reported by Rechavi et al. (4) Further families rated the psychological support in our NBS program studies may provide data to define the reason for these differences very favorably. and their clinical implications, if any. The main limitation of this study is the relatively small With regard to gestational age, median TREC levels in number of newborns screened, although the similarity between our cohort rose significantly from 28 to 32 weeks gestation our results and those reported in larger cohorts supports their in accordance with T-cell maturation in this period, a wider validity. In addition, retrospective data show an incidence of period of time than those reported by other authors (4, 18, 26) SCID in our region similar to that of other Western European (Supplementary Material S4). countries (6, 18) and the United States (5). Overall, there were 30 positive detections in the 129,614 To conclude, TREC quantification in DBS for SCID detection newborns screened (130,903 before applying the exclusion has been satisfactorily implemented in the Catalonian NBS criteria). All were referred to the SCID-CRU per protocol. program. Retesting, requested second samples, and positive Nine patients were considered to have false-positive results due detection rates were optimal with the current algorithm and to initially normal total lymphocyte and CD3+ counts with similar to published data. Our results provide further evidence to normalization of TRECs between 3 and 6 months of life, whereas support the inclusion of SCID in NBS programs in other regions four others had transient lymphopenia at the beginning with and countries. Longer follow-up is needed to define the exact recovery in the following months, and three are currently under incidence of SCID in Catalonia. study [none of the three patients under study met the SCID criteria, as defined by Kwan et al. (2)]. The diagnosis of the T-B-NK+ SCID patient permitted timely DATA AVAILABILITY STATEMENT referral to a specialized treatment center for HSCT, after which T and B lymphocyte counts normalized with fully reconstituted The datasets generated for this study are available on request to function, eliminating the need for IgG supplementation at 1 year the corresponding author. of age. The incidence of SCID found in this study (1 in 130,903) is lower than the reported rates from other areas (2, 4, 24). However, ETHICS STATEMENT we anticipate that a more robust incidence will be established when the population analyzed reaches 200,000 newborns. In The studies involving human participants were reviewed and this line, a retrospective study in our region demonstrated an approved by Comité Ético from Agència de Salut Pública incidence of classically diagnosed SCID of 1 in 57,000 newborns (Catalonia). Written informed consent to participate in this study (unpublished data). In addition to the SCID patient, 13 infants was provided by the participants’ legal guardian/next of kin. with non-SCID TCL were identified, yielding an incidence of 1 in 10,069 newborns (43% positive detections). This is similar to the rate reported by others (31). The diagnostic distribution AUTHOR CONTRIBUTIONS was also comparable. It is worth mentioning that early detection of patients with 22q11.2 deletion syndrome allowed a prompt AA-R, AM-N, and PS-P designed the study. YQ and TC collected intervention to anticipate complications, as reported by our the data and participated in the analytical tools. AA-R and group (Mol Genet Genomic Med, accepted). JM-S organized the database. AA-R and RL-G performed the Of note, one of the strengths of our SCID NBS approach statistical analysis. Results were analyzed and interpreted by is the psychological support offered to the families. A positive AA-R, AR, AM-N, and PS-P. AA-R, AM-N, and PS-P wrote the result in any NBS program is a cause of concern for parents first draft of the manuscript. MG-P, MM-G, JR, RC, SP-G, JG, at this vulnerable time because of an unfamiliarity with these JG-V, and JM-S wrote sections of the manuscript. All authors illnesses, the young age of the patients, and the stress on families contributed to manuscript revision and read and approved the after a birth. Therefore, in neonates initially testing positive, a submitted version. Frontiers in Immunology | www.frontiersin.org 8 October 2019 | Volume 10 | Article 2406 Argudo-Ramírez et al. SCID NBS in Catalonia ACKNOWLEDGMENTS the PID group from the Catalan Societies of Pediatrics and Immunology for their invaluable help to implement SCID NBS in Dr. Marie Audrain (CHU de Nantes, Nantes, France) for Catalonia. We are grateful to the laboratory technicians from the contributing to the development of the algorithm. Mr. Antoni Newborn Screening Laboratory of Hospital Clínic de Barcelona Comin, former Catalan Minister of Health, for his personal and from the Immunology Division of Hospital Universitari implication in the implementation of SCID NBS in Catalonia. Vall d’Hebron. Dr. Rosa Fernández, former Mother and Child Health Division Chief (Health Department) for her support and professionalism with the NBS Program of Catalonia. Mrs. Anna Fabregas, for SUPPLEMENTARY MATERIAL providing psychological support to all patients’s parents with positive results in the SCID NBS screening. Mrs. Marta Parellada, The Supplementary Material for this article can be found for her valuable administrative work. The Catalan Association online at: https://www.frontiersin.org/articles/10.3389/fimmu. for Primary Immune Defects, the BCN-PID Foundation, and 2019.02406/full#supplementary-material REFERENCES 14. Borte S, Wang N, Oskarsdóttir S, von Döbeln U, Hammarström L. 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Verbsky JW, Baker MW, Grossman WJ, Hintermeyer M, Dasu T, Bonacci provided the original author(s) and the copyright owner(s) are credited and that the B, et al. Newborn screening for severe combined immunodeficiency; original publication in this journal is cited, in accordance with accepted academic the Wisconsin experience (2008-2011). J Clin Immunol. (2012) 32:82– practice. No use, distribution or reproduction is permitted which does not comply 8. doi: 10.1007/s10875-011-9609-4 with these terms. Frontiers in Immunology | www.frontiersin.org 10 October 2019 | Volume 10 | Article 2406