Nationwide Pilot Newborn Screening for Spinal Muscular Atrophy and Primary Immunodeficiencies in Greece

1. Introduction

Newborn screening enables the early detection of serious inherited disorders before the onset of clinical manifestations, allowing timely intervention and improving clinical outcomes. This is particularly important for conditions such as Spinal Muscular Atrophy (SMA) and severe T- and B-cell lymphopenias, in which affected newborns may initially be asymptomatic despite the risk of rapid disease progression and irreversible complications [1,2].

SMA is an autosomal recessive neuromuscular disorder caused by biallelic pathogenic variants in the SMN1 (survival motor neuron 1) gene and characterized by progressive loss of spinal motor neurons. Approximately 95–98% of individuals with SMA have a homozygous deletion of exon 7 of SMN1, while the remaining cases are typically caused by a combination of an exon 7 deletion on one allele and a pathogenic sequence variant on the other [3]. The SMN2 gene, a highly homologous paralog of SMN1, produces predominantly non-functional SMN protein, with only a small proportion of its transcripts producing full-length, functional SMN protein; SMN2 copy number is an important modifier of disease severity and an imperfect predictor of clinical phenotype [3,4]. Severe forms present during infancy with profound muscle weakness and progressive respiratory insufficiency and, without effective treatment, lead to early death [3].

The introduction of disease-modifying therapies has substantially changed the prognosis of SMA, with the greatest clinical benefit observed when treatment is initiated pre-symptomatically or very early in the disease course [4,5]. Newborn screening therefore enables identification of affected infants at a stage when rapid diagnostic confirmation and treatment can be initiated, before significant disease progression occurs.

Primary Immunodeficiencies (PIDs), also referred to as inborn errors of immunity, comprise a heterogeneous group of disorders affecting immune system development or function. Severe T- and B-cell lymphopenias, including severe combined immunodeficiency (SCID), are particularly relevant to newborn screening because affected infants may appear clinically well at birth despite a high risk of severe and potentially life-threatening infections [1,2]. T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are circular DNA by-products generated during T- and B-cell development, respectively. Their quantification in newborn screening samples provides an indirect assessment of newly generated T- and B-cell populations and enables the detection of severe T- and/or B-cell lymphopenia [1,2]. Recent population-based experience has demonstrated that combined TREC/KREC screening can identify SCID, XLA, congenital athymia, other clinically significant T-cell deficiencies, and B-cell disorders, while also detecting transient abnormalities requiring careful interpretation [2,6].

The clinical benefits of early diagnosis and the availability of effective interventions including bone marrow transplantation (BMT), cultured thymus tissue implantation (CTTI), enzyme replacement, and gene therapy have led to the expansion of newborn screening programs for SMA and PIDs in an increasing number of countries [1,7]. However, substantial differences remain in their implementation, screening algorithms, referral thresholds, and follow-up pathways. Recent European data demonstrate considerable variation in the implementation of TREC-based screening for SCID and severe T-cell lymphopenia, highlighting the need for harmonized approaches to screening, interpretation, and follow-up [8]. Similarly, recent large-scale experience with combined TREC/KREC screening has demonstrated the feasibility of population-based implementation while emphasizing the importance of appropriate referral and confirmatory pathways [2,6].

In Greece, newborn screening for SMA and severe T- and B-cell lymphopenias was not included in the established national newborn screening program prior to the implementation of the current pilot initiative. In 2025, the pilot implementation of newborn screening for SMA and severe immunodeficiencies was launched within the framework of the National Public Health Prevention Program “Spyros Doxiadis”, with funding from the Greek Recovery and Resilience Facility. The program was designed to support early detection and timely clinical management of affected newborns at a nationwide level [9].

The aim of the present study was to implement and evaluate, at a nationwide level in Greece, newborn screening for SMA and severe T- and B-cell lymphopenias and to assess its feasibility and diagnostic yield in a large population-based cohort. We determined the frequency of screen-positive results, characterized the conditions identified through the screening algorithm, and evaluated the performance of the screening approach in the Greek newborn population. The findings are intended to provide evidence to inform future decisions regarding the potential incorporation of these conditions into the national newborn screening program.

2. Materials and Methods

2.1. Study Population and Sample Collection

Newborns undergoing routine screening through the national newborn screening program were eligible for inclusion in this nationwide pilot screening study. Dried blood spot (DBS) samples routinely collected as part of the national newborn screening process were subsequently used for additional screening for SMA and severe T- and B-cell lymphopenias within the framework of the pilot study. No additional blood collection was required.

For the present analysis, DBS specimens from 29.460 newborns were included. The nationwide newborn screening program officially commenced on 1 May 2026. During the initial implementation phase, approximately 1.000 DBS specimens received through the national newborn screening program were randomly selected and analyzed retrospectively to evaluate the initial performance and workflow of the newly implemented screening process. The remaining specimens were analyzed prospectively following the official commencement of the screening program.

The nationwide pilot program was designed to screen approximately 35.000 newborns by the end of October 2026; thus, the present analysis represents the initial cohort included in the nationwide pilot through 31 August 2026.

2.2. Dried Blood Spot Specimen Collection

Newborn screening specimens were collected as dried blood spots (DBS) on standardized DBS collection cards (Revvity, Massachusetts, USA) in accordance with the applicable newborn screening procedures. For each specimen, a 3.2-mm-diameter DBS punch was automatically transferred into the microtiter plates provided with the screening kit. DBS punching was performed using the Panthera-Puncher™ 9 (Revvity, Massachusetts, USA).

2.3. Multiplex TREC/KREC/SMN1 Newborn Screening Assay

Simultaneous newborn screening for SCID, XLA, and SMA was performed using the commercially available in vitro diagnostic SPOT-it™ TREC, KREC & SMN1 Screening Kit (ImmunoIVD AB, Sweden) for real-time quantitative polymerase chain reaction (qPCR). The assay incorporates standards containing known quantities of the respective molecular targets, allowing semi-quantitative determination of TREC and KREC levels, thus they are measured in copy numbers / punch. In contrast, SMA screening was based on qualitative detection of the SMN1 exon 7 target and is measured by the quantification cycle (Cq).

T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) were used as molecular markers for the assessment of T-cell and B-cell lymphopenia, respectively. For SMA screening, the SMN1 target was used for the qualitative detection of the homozygous deletion of exon 7 of the SMN1 gene. The beta-actin gene (ACTB) was included as an internal control to assess specimen adequacy and amplification performance.

2.4. DNA Extraction and Real-Time qPCR Analysis

All laboratory procedures, including sample preparation, DNA elution, amplification, data analysis, quality control, and result interpretation, were performed in accordance with the manufacturer's validated protocol and Instructions for Use (IFU).

Briefly, following sample preparation, washing, and rehydration, DNA was eluted and transferred to the qPCR plates using centrifugation and plate-stacking technology. The qPCR plates were pre-filled with the required assay reagents, including primers, probes, DNA polymerase, and other components necessary for amplification.

Real-time qPCR amplification and data analysis were performed using a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Applied Biosystems QuantStudio Design and Analysis Software version 2.8.0 (Thermo Fisher Scientific, Waltham, MA, USA). Raw qPCR data were evaluated using the SPOT-it™ Analysis Software provided by ImmunoIVD AB according to the manufacturer's predefined analysis criteria.

2.5. Analytical Quality Control and Assay Validity

Analytical quality control was performed for each assay run by assessing the standard curve parameters, including slope, amplification efficiency, and coefficient of determination (R²), together with the assay-specific control materials. Three control cards, serving as positive controls for the respective assay targets, were included in each analytical run.

At the individual specimen level, ACTB amplification was used as an internal control for specimen adequacy and overall assay performance. Samples or analytical runs that did not meet the predefined validity criteria were considered invalid and were repeated until a valid analytical result was obtained, in accordance with the manufacturer's predefined screening algorithm.

2.6. Screening Cut-Offs and Result Classification

Screening results were classified as within range, out of range, inconclusive, or invalid, as applicable (Table 1).

Following the initial analysis, specimens in which all disease-specific markers were within the predefined reference ranges were classified as negative and required no further analysis. Following the initial analysis, specimens in which all disease-specific markers were within the predefined pathological cut-offs were classified as negative and required no further analysis. Specimens with at least one disease-specific marker outside the predefined pathological cut-off (KREC ≤6 or no amplification, TREC ≤4 or no amplification, or SMN1 ≥32 or no amplification) and an ACTB result within the acceptable range (1.000 ≤ ACTB ≤50.000) were classified as out of range. Specimens with an ACTB result outside the predefined validity range and/or with at least one disease-specific marker outside the predefined validity ranges for negative and out-of-range classification (KREC ≥800, TREC ≥1.200, or SMN1 ≤21), and/or with a deviating amplification curve shape, wereclassified as inconclusive.

2.7. Repeat Testing and Recall Algorithm

Specimens classified as out of range or inconclusive were reanalyzed in duplicate from the same DBS card, resulting in the analysis of three analytical data points per marker, including the initial analysis.

Results were subsequently classified according to the predefined marker-specific decision criteria. A presumptive positive screening result for SMA, SCID, or XLA required at least two of the three measurements for the corresponding molecular marker to be outside the predefined reference range.

Specimens with two of three measurements within the reference range were classified as negative for the respective condition.

A second DBS specimen was requested for all samples yielding a presumptive positive screening result. If neither the criteria for a negative nor those for a presumptive positive result were fulfilled, the triplicate result was classified as inconclusive, and a second DBS specimen was requested. Results obtained from the second DBS card were interpreted according to the same marker-specific decision criteria.

The complete screening workflow and decision algorithm are presented in Supplementary Figure S1.

2.8. Confirmatory Diagnostic Evaluation and Clinical Follow-up

The SPOT-it™ TREC, KREC & SMN1 Screening Kit was used exclusively as a newborn screening assay. An abnormal or presumptive positive screening result was not considered diagnostic and required appropriate confirmatory clinical and laboratory evaluation before a final diagnosis could be established.

Newborns in whom a presumptive positive screening result were referred for condition-specific confirmatory evaluation. For suspected SMA, confirmatory molecular testing was performed by multiplex ligation-dependent probe amplification (MLPA) to determine SMN1 and SMN2 copy numbers using the SALSA MLPA Probemix P021/P060 SMN1/SMN2 (MRC Holland). Newborns in whom a homozygous deletion of SMN1 was confirmed were referred for assessment by a pediatric neurology specialist.

For specimens with out of range TREC and/or KREC screening results, confirmatory immunological evaluation included flow-cytometric assessment of lymphocyte subsets and measurement of immunoglobulin levels. Whole-exome sequencing (WES) was subsequently performed when the findings of the immunological evaluation indicated the need for further genetic investigation.

3. Results

During the study period, 29.460 newborns were screened, with one dried blood spot (DBS) card analyzed for each newborn as part of the initial screening round. Of these, 27.292 newborns were immediately classified as negative for all three screening targets (SMN1, TREC, and KREC) and required no further analysis. The remaining 2.168 newborns (7,36%) yielded an out-of-range or inconclusive result for at least one screening marker and underwent further assessment according to the predefined screening algorithm. Among these, 2.076 were classified as inconclusive, including 55 with invalid results, and 92 as out of range.

The laboratory turnaround time, defined as the interval from receipt of the DBS card at the screening laboratory to availability of the initial screening result, was generally 2–3 days.

All 2.168 newborns with an out-of-range or inconclusive initial result underwent duplicate retesting from the same DBS card. Following retesting, 1.687 newborns (77,8%) were resolved, whereas 481 (22,2%) required collection of a second DBS card. Thus, 7,36% of the screened newborns required duplicate retesting, while 1,63% required a repeat DBS sample.

Of the 481 newborns for whom a second DBS card was requested, 469 second DBS cards were received, while 12 were not received, including 9 newborns who were lost to follow-up and 3 who died before repeat sampling. Among the 469 second DBS cards received, 446 newborns were classified as negative, 13 remained inconclusive, 4 were referred for genetic and/or immunological confirmatory evaluation, 2 remained under immunological follow-up, and 4 demonstrated recovery of previously out-of-range results on repeat testing. Together, these results accounted for all 469 second DBS cards received. The complete screening workflow and observed outcomes are shown in Supplementary Figure S1.

The results requiring further clinical and/or laboratory evaluation are summarized in Table 2. Detailed longitudinal immunological findings for the four most clinically significant cases from TREC/KREC screening are presented in Supplementary Table S1.

Case 1 was identified based on a presumptive positive SMN1 screening result (Supplementary Figure S2). Genetic confirmation by MLPA demonstrated a homozygous deletion of the SMN1 gene and three copies of SMN2. Based on the molecular findings, the patient would be expected to have a SMA phenotype, most consistent with SMA type 2 or 3. The patient underwent SMN1 gene-replacement therapy without complications and remains clinically well at 3 months of age.

Case 2 presented with generalized hypotonia, decreased antigravity movements, and absent tendon reflexes soon after birth. The newborn had been included among the DBS specimens collected in April, before the formal commencement of the screening program, and subsequently analyzed retrospectively during the initial evaluation of the screening implementation. Genetic confirmation by MLPA demonstrated a homozygous deletion of SMN1 and two copies of SMN2, consistent with a diagnosis of SMA type 1. While awaiting the genetic report, the newborn was referred by the family pediatrician on the 32nd day of life to a Pediatric Neurology Unit in close proximity to the family’s residence for further clinical evaluation and management. Following confirmation of the genetic findings, the patient underwent SMN1 gene-replacement therapy on day 49 of life without complications and remains clinically well, with definite clinical improvement at 5 months of age.

Case 3 was identified following an out of range TREC result, while SMN1 amplification was detected as normal and KREC values were within the normal range (Supplementary Figure S2). The neonate was referred for urgent immunological evaluation. Flow cytometric analysis confirmed an absence of detectable T cells, including naïve T cells and recent thymic emigrants (RTEs), consistent with severe T-cell lymphopenia (Supplementary Table S1).

Further genetic investigation was performed using WES. No pathogenic or likely pathogenic variants were identified in genes relevant to the patient’s clinical phenotype. However, structural variant meta-analysis of the WES data identified a heterozygous deletion of approximately 2.5 Mb involving the 22q11.2 region. The finding was considered clinically significant and potentially explanatory of the patient’s immunological phenotype. The deletion was subsequently confirmed by molecular karyotyping, confirming the diagnosis of 22q11.2 deletion syndrome (complete DiGeorge syndrome).

Clinical evaluation revealed no evidence of congenital heart disease. Serum calcium and parathyroid hormone levels were within the normal range, and no dysmorphic features were observed. Given the severe T-cell lymphopenia, strict isolation, antimicrobial and antifungal medication as well as Ig replacement were initiated to prevent life threatening infections while planned definitive CCTI.

Case 4 was a neonate born at 24 weeks of gestation with a prenatally diagnosed 22q11.2 deletion syndrome. A DBS sample was collected shortly after admission to the neonatal intensive care unit (NICU). The screening result was classified as inconclusive according to the ImmunoIVD diagnostic algorithm because of relatively low amplification across all assay targets. TREC amplification was detectable but markedly reduced relative to KREC, SMN1, and ACTB, with TREC values of 3,687, 9,77 and 10,101 copies/punch, respectively. KREC, SMN1, and ACTB were also assessed.

The infant died several hours after birth in the setting of extreme prematurity and respiratory distress. Preterm delivery was attributed to maternal chorioamnionitis due to Escherichia coli. Given the extremely low gestational age and the short postnatal survival period, further immunological and clinical evaluation could not be performed.

Case 5 was a late preterm neonate born at 36 weeks of gestation whose initial DBS screening revealed low TREC levels. When the family was contacted to request a second DBS card, the infant was reported to be hospitalized in the NICU with congenital heart disease, hypertelorism, iris coloboma, and external ear anomalies. Given the clinical findings, the neonate was referred by the Neonatal Unit to the Immunology Unit for immunological evaluation, which was performed concurrently with the screening follow-up and showed borderline T-lymphocyte levels (Supplementary Table S1).

Subsequent WES identified the pathogenic variant CHD7:c.7879C>T in a de novo heterozygous state. The combination of the clinical, immunological, and genetic findings was consistent with a diagnosis of CHARGE syndrome.

Regarding TREC screening, two additional neonates presented with TREC values below the established screening cut-off in the context of prematurity.

Case 6 was born at 26 weeks of gestation, with an extremely low birth weight of 580 g, while Case 7 was born at 32 weeks of gestation. In both cases, the initially low TREC levels were interpreted in the context of prematurity. Repeat screening using a second DBS card, collected approximately 15 days after birth, demonstrated an increasing trend in TREC levels in both neonates. Subsequent measurements confirmed recovery of TREC levels.

In addition, several neonates with low or undetectable KREC levels were identified and underwent further evaluation or follow-up. Case 8 was identified based on initially low KREC levels on neonatal screening and was referred by the screening team to the Immunology Unit for further immunological evaluation. The immunological assessment confirmed the findings of the initial screening (Supplementary Table S1). However, subsequent measurements demonstrated a gradual recovery of KREC levels over time. A detailed family history revealed maternal treatment during pregnancy with methylprednisolone, acetylsalicylic acid, hydroxychloroquine, and azithromycin in the context of systematic lupus erythematosus. The infant remains under clinical and immunological follow-up.

Additional neonates with reduced or undetectable KREC levels were also identified during screening. One infant (Case 9), born at 30 weeks of gestation with a birth weight of 1.345 g, subsequently died during the neonatal period, with prematurity considered a likely contributing factor. Another neonate (Case 10), born at 38 weeks of gestation with a birth weight of 2.140 g, developed staphylococcal sepsis one week after birth and subsequently died. Both infants were lost to further immunological follow-up.

Two additional neonates demonstrated recovery of KREC levels on repeat testing. One infant (Case 11), born at 39⁺³ weeks of gestation with a birth weight of 3.554 g, demonstrated subsequent recovery of KREC levels. Another infant (Case 12), born at 38 weeks of gestation with a birth weight of 2.820 g, was diagnosed with esophageal atresia and underwent surgical intervention; KREC levels subsequently recovered.

An additional neonate (Case 13), born at 38 weeks of gestation with a birth weight of 2.600 g, presented with a low KREC value in the initial DBS sample. Immunological evaluation revealed low B-cell numbers (Supplementary Table S1). The infant remained under close clinical and immunological follow-up, with subsequent recovery of both KREC levels and B-cell numbers approximately one month after birth.

4. Discussion

This nationwide pilot study provides the first nationwide experience in Greece with the implementation of combined newborn screening for SMA and severe T- and B-cell lymphopenias within the Greek national newborn screening infrastructure. Rather than addressing the feasibility of combining these screening targets, which has already been demonstrated in other population-based programs, our findings provide insights into the practical performance of such an approach when implemented across a geographically distributed national screening system.

An important consideration in the implementation of newborn screening is the time required to reach a final screening classification. In our program, the routine laboratory turnaround time, defined as the interval from receipt of the DBS card at the screening laboratory to availability of the initial screening result, was generally 2–3 days. However, the overall time to final screening classification was influenced by the need for repeat testing, repeat DBS collection, and, in presumptive-positive cases, confirmatory diagnostic evaluation. In one case, the time to final classification extended to 25 days. This was attributable to the retrospective analysis of a DBS specimen collected before the formal initiation of the screening program and therefore does not reflect the routine laboratory turnaround time. Nevertheless, this case illustrates that, in real-world implementation, additional laboratory investigations, repeat sampling, and confirmatory testing may substantially influence the time required for final resolution of a screening result. These factors should therefore be considered when evaluating the operational performance of integrated newborn screening programs.

In our cohort, 7,36% of newborns required repeat testing from the initial DBS card, while 1,63% required collection of a second DBS specimen. These rates are higher than those reported in several established TREC/KREC-based newborn screening programs, although comparable repeat-testing rates have been described in some pilot settings. In the 2026 nationwide Japanese program, 0,25% of newborns required a second DBS specimen, while in the five-year combined SMA/SCID screening program in Liguria, Italy, 0,12% required a second DBS after duplicate retesting of the initial sample [10,11]. By contrast, a TREC/KREC pilot study using a higher cutoff reported a repeat-testing rate of 7,6% and a repeat-sampling rate of 0,9%, which decreased to 4,1% and 0,5%, respectively, after lowering the cutoff [12]. Direct comparisons, however, are limited by differences in assay platforms, cutoff values, specimen-handling procedures, and screening algorithms. In our program, the predefined algorithm required duplicate retesting of all specimens initially classified as out of range or inconclusive, which may have contributed to the observed repeat-testing rate.

An additional consideration is the heterogeneity of DBS collection across a nationwide screening network, DBS specimens were collected at multiple sites across Greece using different sampling practices, including direct capillary blood collection and transfer of blood obtained using microhematocrit capillary tubes [13]. Such geographic and procedural variability may affect specimen quality, blood volume, and spot uniformity and may consequently contribute to the need for repeat analysis or recollection. Although specimen-quality parameters were not prospectively recorded and individual repeat-testing events could not be directly attributed to specific pre-analytical factors, this heterogeneity represents an important consideration when interpreting the observed repeat rates. Standardization of DBS collection and ongoing quality monitoring may therefore be important for optimizing the performance of nationwide screening programs.

The identification of two newborns with SMA among approximately 30.000 screened infants highlights the clinical value of incorporating SMN1 analysis into newborn screening. In both cases, the diagnosis was confirmed by MLPA, demonstrating homozygous SMN1 deletion with three and two SMN2 copies, respectively [3,4]. The identification of these cases through newborn screening enabled referral for specialized clinical evaluation and facilitated early diagnostic confirmation. The different SMN2 copy numbers provided clinically relevant information for prognostic assessment and were an important consideration in subsequent treatment planning [5].

Concerning T- and B-cell lymphopenias, our findings are consistent with previous studies showing that TREC-based newborn screening can identify significant T-cell lymphopenia beyond classical SCID [14,15]. Although no cases of SCID were detected in our cohort, abnormal TREC results led to the identification of clinically relevant T-cell abnormalities in two neonates with syndromic conditions. One neonate with persistently undetectable TREC levels was diagnosed with 22q11.2 deletion syndrome and profound T-cell lymphopenia, while another neonate with low TREC levels was subsequently diagnosed with CHARGE syndrome and demonstrated borderline T-lymphocyte levels on immunological evaluation. These cases illustrate the broader clinical utility of TREC screening in identifying clinically relevant T-cell abnormalities and underscore the importance of interpreting abnormal results in the context of the underlying clinical and genetic findings [16].

The neonate with 22q11.2 deletion syndrome born at 24 weeks of gestation illustrates the complexity of interpreting TREC-based screening in the setting of extreme prematurity. In this case, the low TREC levels may have reflected the combined effects of extreme prematurity and the underlying genetic condition, which may itself be associated with impaired thymic development. However, the infant’s very early death precluded confirmatory immunological evaluation, preventing definitive assessment of the relative contribution of these factors.

The transiently low TREC levels observed in several neonates in our cohort are consistent with the findings of Soomann et al., who reported an association between transiently abnormal TREC results and prematurity and low birth weight [2]. Similarly, several preterm infants in our cohort demonstrated recovery of TREC levels on repeat DBS sampling, supporting the potential transient nature of these findings and the influence of developmental immaturity on thymic output. These observations further emphasize the importance of considering gestational age and clinical context when interpreting low TREC results.

Of particular note, KREC findings in our cohort highlight the importance of clinical context in interpreting abnormal results. One infant with persistently low KREC levels on initial follow-up had been exposed to maternal immunosuppressive therapy during pregnancy, a potential factor that may have contributed to the observed finding, as previously suggested by Soomann et al, [2]. A second infant with reduced KREC levels subsequently developed severe staphylococcal sepsis and died during the neonatal period, although a causal relationship between the abnormal KREC result and the subsequent clinical course cannot be established. These observations support the value of integrating maternal history and the clinical condition of the newborn into the interpretation of KREC screening results.

A major strength of this study is its nationwide scope and implementation, providing real-world experience with the implementation of combined SMA, TREC, and KREC newborn screening within the Greek national newborn screening infrastructure. The inclusion of 29.460 newborns allowed evaluation of the screening workflow under routine operational conditions across a geographically distributed population and provided an opportunity to characterize both clinically significant findings and transient or inconclusive results. The use of a single multiplex assay for simultaneous assessment of the three screening targets also represents an important practical advantage, facilitating integration into the existing newborn screening workflow.

Several limitations should nevertheless be taken into consideration. First, this was an observational pilot implementation, and the study was not designed to determine the diagnostic sensitivity or specificity of the screening strategy for each individual condition. Second, the number of confirmed cases was limited, and the screening period was relatively short; therefore, the present cohort does not allow a robust estimate of the population frequency of SMA in Greece. Nevertheless, the observed number of SMA cases may be considered in the context of previously reported national epidemiological data [17]. Third, detailed pre-analytical information, including specimen-quality parameters and the specific circumstances of individual sample collections, was not prospectively recorded, limiting assessment of the factors contributing to repeat testing and repeat sampling. In addition, some infants with abnormal screening results could not undergo complete confirmatory evaluation because of clinical circumstances or loss to follow-up. Finally, the study represents the experience of a single national screening laboratory, and the findings may not be directly generalizable to other screening settings using different assay platforms, cutoff values, or screening algorithms.

Overall, the findings from this cohort demonstrate the potential clinical value of combined newborn screening for SMA, T-cell, and B-cell lymphopenias, SMN1 screening enabled the identification of newborns with SMA, while TREC and KREC screening identified clinically relevant immune abnormalities, including syndromic conditions, as well as transiently low marker levels associated with prematurity and other clinical or maternal factors. The combination of repeat DBS testing, appropriate confirmatory evaluation, and clinical follow-up was important for distinguishing infants requiring further investigation from those whose abnormal screening results subsequently resolved. Persistently undetectable or markedly reduced TREC or KREC levels should prompt timely specialist evaluation, whereas recovery on repeat testing may support a transient process, particularly when considered in the context of gestational age and the infant’s clinical condition.

5. Conclusions

This study represents an important step in the development of newborn screening program in Greece, describing the implementation of combined SMA, TREC, and KREC screening for the first time at the national level. The introduction of these screening targets represents an important expansion of the Greek newborn screening program and contributes to its alignment with evolving international practices, in which screening for SMA and severe T- and B-cell lymphopenias has been increasingly incorporated into national and regional programs. Although implementation has occurred later than in several other countries, the establishment of this program provides an important framework for the early identification of infants with SMA and clinically significant lymphocyte abnormalities. Our findings also highlight the importance of standardized screening procedures, appropriate interpretation of abnormal results, and timely confirmatory clinical, immunological, and genetic evaluation. Continued implementation and systematic prospective evaluation will be essential to further optimize the screening algorithm, assess its long-term clinical impact, and further inform the development of newborn screening in Greece.