Newborn screening (NBS) for Severe Combined Immunodeficiency (SCID) rapidly expanded across the United States (US) since 2010. By December 2018, all 50 states had begun screening for SCID (1). SCID is not easily recognizable clinically at birth and is near uniformly fatal by 1 year of age without early treatment. Furthermore, immune function can be restored with life-saving hematopoietic cell transplantation (HCT), and survival is superior if HCT is performed in the first few months of life (2, 3). Finally, the absence or marked reduction of T cell receptor excision circles (TRECs) is highly associated with SCID, and TRECs are readily identifiable in the newborn dried blood spots (DBS) (4) of healthy newborns (5) thus SCID is particularly well suited for population-based NBS. In May of 2010, following reports of findings from earlier pilot studies in Wisconsin, the Navajo Nation, and our own statewide pilot SCID NBS program in Massachusetts, SCID was added to the US recommended uniform screening panel of conditions (6–8). This report presents a summary of findings from Massachusetts’ first 10 years of SCID NBS experience, capitalizing on our ongoing data analyses for evidence-based quality improvement modifications to interpretation and notification protocols. We present our review of the diagnostic workup, in which we analyzed the relative value of flow cytometry and lymphocyte proliferation measurements for predicting a diagnosis of SCID or another genetic form of immunodeficiency. Additionally, we present TREC values linked to infants’ gestational ages. We report the incidence of SCID in Massachusetts, the outcomes of definitive cellular therapy for the identified infants, and the diagnoses of additional infants with other conditions treated with cellular therapy.

The New England Newborn Screening Program (NENSP) developed and validated a high-throughput multiplex qPCR assay quantifying both the TREC analyte and an internal control (a single copy gene, RNaseP) in DNA extracted from a single DBS punch (9). The Commonwealth of Massachusetts authorized a statewide SCID NBS pilot that was initiated February 1, 2009 using a verbal consent protocol (105CMR270.010) yielding 98% enrollment; in 2016 SCID NBS became mandatory. Infants whose specimens showed low or absent TREC values were considered to have out-of-range SCID NBS results that required either a repeat NBS specimen or diagnostic testing of lymphocyte subsets with specified flow cytometry.

We routinely evaluated screening data and diagnostic outcomes using a baby-specific interpretation and reporting algorithm, that interpreted each individual test in the context of all other results from that specific baby. Our goal was to reduce the burden of positive screens while maintaining appropriate sensitivity. We developed a streamlined interpretation and notification protocol that discriminated between low and undetectable TREC values and that utilized evaluation of serial TREC values in neonatal intensive care unit (NICU) and non-NICU populations. Based on accruing data from the first two years of screening, we made three key evidence-based changes to our notification recommendations: 1) Initially, any infant with an out-of-range TREC result on any specimen (initial or repeat) was referred for lymphocyte subset testing. To avoid overwhelming primary care and NICU providers, families, and the small number of flow cytometry laboratories that met criteria to perform specialized pediatric diagnostic testing, we used data accrued among 16 infants referred during the first month of screening and changed the recommended action so that only infants with at least two out-of-range SCID NBS results would be referred for diagnostic testing by flow cytometry. 2) After several months of screening (~110,000 infants), we observed that an undetectable TREC result on initial NBS specimens was both very infrequent (at the time, 0.01% of all infants screened; 4% of out-of-range screening results) and consistent with results of retrieved specimens tested from well-characterized SCID patients during our testing validation. We thus modified our interpretation and notification protocol to recommend immediate lymphocyte subset testing for any infant with an undetectable TREC result on an initial NBS specimen. 3) Simultaneously, we determined that 66 infants with serial NBS specimens who had at least one normal TREC result among multiple out-of-range TREC results had diagnostic results inconsistent with SCID. All 66 of these infants had prolonged NICU courses, often with complications including, but not limited to, extreme prematurity, cardiac defects, chylothorax, hydrops, gastrointestinal complications, and various genetic syndromes. We therefore modified our recommended-actions algorithm to allow us to release from follow up those infants who had serial specimens that included at least one normal TREC value.

To investigate the TREC values for premature infants, we evaluated data that were linked to infant gestational age at the time of specimen collection. For a subset of all specimens (infants from a three-month period in 2015), gestational age at birth was manually entered into a database. The difference between each infant’s date of birth and date of specimen collection was then added to the gestational age at birth in order to calculate each infant’s gestational age at the time of specimen collection.

Our current criteria for referral stipulates that infants are referred for testing of lymphocyte subsets by flow cytometry if they have undetectable TREC values on an initial NBS specimen or if they have out-of-range TREC values (<252 copies/ μL) on two serial specimens in the absence of a normal result. All infants requiring flow cytometry were sent for phlebotomy at one of multiple sites with a specialized requisition for lymphocyte subset testing that would occur within 24 hours. Diagnostic results were reported back to the NENSP for centralized tracking. Reference ranges for percent and absolute counts of lymphocyte populations were adapted from Shearer et al (10) and were standardized across the three sites that meet criteria for performing the testing. The minimal consensus panel included flow cytometry detecting surface expression of CD3, CD4, CD8, CD16 and/or CD56, CD19, and any of the following: CD45RA, CD45RO, CD62L, CD11a dim, CD31(9).

Normal flow cytometry was defined as ≥2500 T cells/μL (based on Shearer et al) and CD4 T cells >50% naïve and CD8 T cells >50% naïve. T-cell lymphopenia (TCL) was defined as having a CD3 <2500 cells/μL. The diagnosis of SCID was made according to criteria of the Primary Immune Deficiency Treatment Consortium (PIDTC), including typical SCID, leaky SCID, and Omenn syndrome (10). Other patients with TCL who did not meet PIDTC criteria for SCID but who had opportunistic infections, hypogammaglobulinemia, abnormal proliferation to phytohemagglutinin (PHA), poor antigen-specific T cell proliferation (e.g., tetanus and candida), and/or poor vaccine response were defined as combined immunodeficiency (CID). We calculated the risk of SCID or TCL and 95% confidence intervals.

Pilot newborn screening programs are typically implemented with conservative follow-up protocols that are later modified as screening and follow up data are accrued. Early in our screening experience as 1 of only 2 states conducting pilots of SCID screening, we pioneered two seminal interpretation and notification modifications based on our own empiric data to improve the overall efficiency and clinical effectiveness of the screening program. We modified our follow-up algorithm to require immediate lymphocyte subset testing for infants with an undetectable TREC result on an initial NBS specimen. Furthermore, we modified our recommendations such that infants with an out-of-range result and an independent specimen that showed a normal TREC result would not be referred for clinical evaluation, provided that the healthcare providers had no clinical concerns for SCID. The changes we made to our interpretation and notification algorithms ultimately decreased the number of infants requiring any further testing (by repeat NBS or flow cytometry) by 20%.

All NBS programs recognize that both NICU status and low gestational age yield a higher frequency of abnormal NBS results for many NBS conditions, and SCID is no exception to this. Eighty-three percent of infants with out-of-range SCID NBS results and 74% of infants prompting referral for lymphocyte subset testing were in a NICU. While NICU infants have a much higher rate of positive screening results compared to non-NICU infants, we believe that the NICU population, especially as it relates to prematurity, is at no lesser risk of having SCID than the general population. In some cases, NICU status might be a risk factor for both SCID and other disorders associated with TCL that might be identified by the screen. In fact, of the 9 Massachusetts infants identified with SCID, two were NICU infants – one who passed through the NICU and one who was seriously ill. The latter infant presented with MIA, was being evaluated for Cystic Fibrosis (CF) (in-range CF NBS results) (12) and had a prolonged and difficult NICU course. SCID has been previously reported to be associated with MIA (13, 14), and this case demonstrates the importance of compliance with NBS follow up recommendations in NICU infants. Our follow up algorithm minimizes the burden on NICU teams and primary care providers while still recognizing the substantially increased risk of SCID in all infants with positive screening results.

While infants born prior to 37 weeks gestation had a lower median TREC value than those born at term, the median TREC value in premature infants was ~4x our pre-determined cutoff value for low TREC and the vast majority (98%) of premature infants had normal screening results. As expected, the frequency of normal NBS decreased from 99% (836/845) for specimens of infants aged 28 – 37 weeks to 81% (34/42) for infants aged < 28 weeks. Lymphopenia is common in preterm infants and affects all lymphocyte subsets, CD4⁺ and CD8⁺ T cells, B cells and NK cells, with infants at the lowest gestational age demonstrating the most severe lymphopenia (15). Premature (and NICU) infants routinely experience stressful stimuli that induce endogenous cortisol production (16). Both endogenous and exogenous corticosteroids reduce lymphocyte number and function (17, 18). Decreased concentration of IL-7 and reduced expression of IL-7 receptor in T cells of low gestational age infants has been proposed as another potential mechanism of the T cell lymphopenia (15). We nevertheless contend that the risk of SCID among premature and NICU infants with out-of-range TREC results is high enough to warrant the same timely follow up as infants born at term, and that follow up recommendations must be the same for all infants regardless of NICU status or gestational age. The algorithm, which selects urgent (undetectable TREC) and repeatedly abnormal TREC values, minimizes referral. Abnormal TREC results warrant attention in all infants.

With any screening test, there is cost to false positive results – in dollars, utilization of services, and patient/family anxiety. We therefore considered the impact of our cutoff for immunology referral of 2500 T cells/μL, which is higher than that of some other NBS programs, which use cutoffs of 1500 cells/μL. Of the 162 infants we identified with TCL, 44 had T cell counts between 1500 cells/μL and 2499 cells/μL, corresponding to an additional 4.4 cases per year referred for clinical evaluation. Of these 44 infants evaluated by immunologists, 36 did not have any other defined diagnosis explaining TCL (no cases of SCID, leaky SCID, or a monogenic CID), while the remaining 8 infants (18%) were found to have partial DGS. In 3 of these 8 cases, the diagnosis of DGS was made based on NBS (rather than congenital heart disease, other congenital anomalies, or hypocalcemia). How one weighs the benefit of making these diagnoses against the potential negative impact to patients and families who would otherwise not have been evaluated can be debated; for now, we plan to continue to evaluate patients with our current cutoff.

We examined the utility of naïve and memory T cell phenotyping as part of the flow cytometry panel. Over the 10-year period, measurement of naïve CD4 T cell percentage did not lead to additional patient referral for formal immunology evaluation beyond absolute T cell count alone. However, our retrospective review indicates that patients with naïve CD4 T cells <50% were at far greater risk of having SCID or CID compared to those infants with naïve CD4 T cells >50% (relative risk 16, 95% CI: 5.8–46.1). We believe naïve T cell phenotyping therefore remains an important tool for risk stratification, along with absolute T cell counts and clinical history.

Massachusetts ten-year summary data revealed an incidence of SCID (1/80,000, [95% CI 1/48,000–1/240,000]) comparable to that observed in other states (19–23) and consistent with expectations for a rare disorder in a small screened population. We ensured quality data by a strict application of the case definition based on PIDTC criteria and statewide surveillance for potential misses through the MA SCID NBS Working Group, which has broad, statewide, multi-institutional, multidisciplinary representation, and no infants or children with false negative SCID NBS results have come to attention. Two children diagnosed with ZAP70 deficiency had normal TRECs at birth and low TRECs at the time of the diagnostic workup (at ages ~9 months and ~18 months) but this is expected (24–26).

Although our reported SCID incidence of 1/80,000 was comparable to the incidence of 1/125,000 [95% CI: 1/72,917–1/437,500] we observed in the pre-NBS era (7 known SCID infants born in Massachusetts between 1998–2008), the outcomes of the infants were different. All 7 were diagnosed as a result of opportunistic infection, and 2 had active infection at the time of HCT. One died before HCT, 2 died after HCT, and so only 4 of 7 survived (Table E1). In contrast, only 1 of the 9 infants identified by NBS had active infection and all 9 are alive post-transplant with excellent immune outcomes, a difference in survival which trends towards but does not meet significance (Fisher’s exact test, p=0.0625). We note that the median age at transplant was substantially lower, 6.5 months before NBS versus 3.4 months after NBS (Mann-Whitney test, p=0.0174). We are optimistic that the improvements we observed are likely to bear out with more years of screening. We attribute a good portion of our program’s success to effective coordination of screening, clinical follow-up, and treatment by the highly collaborative Massachusetts SCID NBS Working Group. Approximately 15% of 184 infants with lymphocyte subset data available were evaluated at more than 1 institution. The integration of NENSP, flow cytometry labs, and clinical experts in multiple disciplines is of vital importance for the success of SCID NBS.