Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Pediatric Allergy and Immunology (2016): January 21 DOI: 10.1111/pai.12501 Copyright ©: 2016 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is the peer reviewed version of the following article: de Felipe B, Olbrich P, Lucenas JM, Delgado-Pecellin C, Pavon-Delgado A, Marquez J, Salamanca C, Soler-Palacin P, Gonzalez-Granado LI, Antolin LF, Borte S, Neth O. Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville. Pediatr Allergy Immunol. 2016 Feb;27(1):70-7. doi: 10.1111/pai.12501. Epub 2015 Nov 23. PMID: 26498110, which has been published in final form at https://doi.org/10.1111/pai.12501. 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For Peer Review Prospective neonatal s creening for severe T - and B - lymphocyte deficiencies in Seville Journal: Pediatric Allergy and Immunology Manuscript ID PAI-15-O-0258.R1 Manuscript Type: Original Date Submitted by the Author: 06-Oct-2015 Complete List of Authors: de Felipe, Beatriz; Instituto de Investigación Biomedica Sevilla (IBiS), Pediatrics Olbrich, Peter; Hospital Infantil Universitario Virgen del Rocío, Infectious Diseases and Immunopathologies; Instituto de Investigación Biomedica Sevilla (IBiS), Pediatrics Lucenas, Jose; Hospital Universitario Virgen del Rocio, Immunology Delgado-Pecellin, Carmen; Hospital Universitario Virgen del Rocio, Departamento de Bioquímica Clínica Pavon-Delgado, Antonio; Hospital Infantil Universitario Virgen del Rocío, Neonatology Marquez-Fernandez, Josefina; Hospital Universitario de Valme, Neonatology Salamanca, Carmen; Hospital Universitario Virgen de Macarena, Neonatology Soler-Palacín, Pere; Hospital Universitari Vall d'Hebron. Institut de Recerca Vall d'Hebron. Universitat Autònoma de Barcelona, Pediatric Infectious Diseasesm and Immunodeficiencies Unit Gonzalez Granado, Luis; Division of Immune Deficiency, Department of Pediatrics, Hospital 12 de Octubre, Ferreras Antolin, Laura; Hospital Universitario Regional de Málaga, Departamento de Pediatría, Borte, Stephan; Hospital St Georg gGmbH , Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies; University of Leipzig, Translational Centre for Regenerative Medicine(TRM); Karolinska Institutet, Karolinska University Hospital Huddinge, Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine Neth, Olaf; Hospital Infantil Universitario Virgen del Rocío, Infectious Diseases and Immunopathologies; Instituto de Investigación Biomedica Sevilla (IBiS), Pediatrics Topics of paper: immunodeficiency: primary immunodeficiencies, methods: lymphocytes, methods: T cells, public health: national programs, epidemiology: Prevalence, IMMUNODEFICIENCY Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Page 1 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Prospective neonatal screening for severe T- and B- lymphocyte deficiencies using TRECs/KRECs/ACTB real time PCR in Spain Beatriz de Felipe 1 *, Peter Olbrich 1 *, José Manuel Lucenas 2 , Carmen Delgado-Pecellin 3 , Antonio Pavon-Delgado 4 , Josefina Marquez 5 , Carmen Salamanca 6 , Pere Soler-Palacin 7 , Luis Ignacio Gonzalez-Granado 8 , Laura Ferreras Antolin 9 , Stephan Borte 10-12 , Olaf Neth 1 ** 1 Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Spain 2 Unidad de Inmunología, Hospital Universitario Virgen del Rocío, Sevilla, Spain 3 Unidad de Metabolopatías, Hospital Universitario Virgen del Rocío, Sevilla, Spain 4 Unidad de Neonatología, Hospital Universitario Virgen del Rocío, Sevilla, Spain 5 Unidad de Pediatría, Hospital Virgen de Valme, Sevilla, Spain 6 Unidad de Neonatología, Hospital Universitario Virgen de Macarena, Sevilla, Spain 7 Pediatric Infectious Diseases and Immunodeficiencies Unit. Hospital Universitari Vall d'Hebron, Institut de Recerca Vall d'Hebron. Universitat Autònoma de Barcelona (UAB), Spain 8 Division of Immune Deficiency, Department of Pediatrics, Hospital 12 de Octubre, Madrid, Spain 9 Unidad de Enfermedades Infecciosas e Inmunodeficiencias. UGP de Pediatría. Hospital Materno-Infantil. Hospital Regional Universitario de Málaga, Spain 10 Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. Page 2 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 11 Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany. 12 ImmunoDeficiencyCenter Leipzig at Hospital St Georg gGmbH Leipzig, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Leipzig, Germany. *shared 1 st author, **corresponding author Corresponding author: Olaf Neth Unidad de Enfermedades Infecciosas e Inmunopatologias, Hospital Infantil Virgen del Rocio, Instituto de Biomedicina de Sevilla, Av. Manuel Siurot, s/n. 41013, Sevilla, Spain. Phone: +34 622488740; Fax: +34-955012991; e-mail: [email protected] Funding resources: This work was funded by - Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla, Hospital Universitario Virgen Del Rocío (FISEVI, “Scott Family Funding”) - Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III (PI13/01104) - Ayudas para contratos de formación en Investigación Rio Hortega. Instituto de Salud Carlos III. Disclosures: We hereby verify that the manuscript has not been submitted or accepted elsewhere. All authors have contributed to, seen, and approved the final, submitted version of the manuscript. The authors have no conflicts of interest to disclose. Page 3 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Running title: Newborn screening, primary immunodeficiencies, TRECS, KRECS Word count: 2500 Number of tables: 2 Number of figures: 1 Page 4 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Abstract Background: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected children. T-cell-receptor-excision circles (TRECs) and kappa-deleting- recombination-excision circles (KRECs) determination from dried blood spots (DBS) identify neonates with severe T- and/or B-lymphopenia. No prospective data exist of the impact of gestational age (GA) and birth weight (BW) on TRECs and KRECs values. Methods: TRECs and KRECs determination using triplex RT-PCR (TRECS-KRECS-β-actin- Assay) from prospectively collected DBS between 02/2014 and 02/2015 in three hospitals in Seville, Spain. Cut-off levels were TRECs<6/punch, KRECs<4/punch and -β- actin>700/punch. Internal (SCID, XLA, ataxia telangiectasia) and external controls (NBS quality assurance program, CDC) were included. Results: A total of 5160 samples were tested. 77 samples (1.5%) were insufficient (β-actin<700 copies/punch) for analysis. Preterm neonates (GA<37 weeks) and neonates with a BW<2500g showed significantly lower TRECs and KRECs levels (p<0.001). Due to false positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n=1; TRECs 1/KRECs 4); extreme prematurity (n=2; TRECs 0/KRECs 0 and TRECs 1/KRECs 20 copies/punch); neonates born to mothers receiving azathioprine during pregnancy (n=2; TRECs 92/KRECs 1 and TRECs 154/KRECs 3 copies/punch). All internal and external controls were correctly identified. Conclusions: TRECS-KRECS--β-actin-Assay correctly identifies T- and B-cell lymphopenias. Prematurity and low BW is associated with lower TREC and KREC levels. Extreme prematurity and maternal immune suppressive therapy may be a cause for Page 5 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved. Page 6 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Introduction Severe T- and/or B-cell lymphopenias in the first days of life may indicate congenital immune defects such as X-linked agammaglobulinemia (XLA) and severe combined immunodeficiency (SCID) [1]. Although SCID and XLA belong to the group of rare diseases, their incidences of 1:35,000 to 1:100,000 live births is still considerably high given their severity [2,3]. The common lack of a suggestive family history and the absence of specific clinical symptoms in the first weeks of life often lead to a delayed diagnosis, associated with prolonged intensive care treatment, high morbidity and mortality [4-6]. To date, no European country performs newborn screening (NBS) for primary immunodeficiencies (PIDs) routinely, despite meeting most of the established screening criteria, including the existence of relatively non-invasive screening methodology, proven benefit of early diagnosis, presence of an initial asymptomatic period, high morbidity and mortality, availability of curative or effective therapeutic options and a favourable cost-benefit analysis [7-9]. In contrast to most of the diseases included in the routine NBS, curative treatment approaches for SCID patients exist in form of hematopoietic stem cell transplantation (HSCT) or gene therapy [10,11]. Furthermore, early diagnosis avoids preventable iatrogenic events related to live vaccines such as rotavirus [12,13]. In patients with congenital agammaglobulinemias, prompt diagnosis and immunoglobulin substitution have shown to improve overall outcomes [14]. The quantification of two circular DNA fragments (T-cell-receptor-excision circles, TRECs and kappa-deleting-recombination-excision circles, KRECs) in dried blood spots (DBS) and tandem mass spectrometry (TMS) for enzyme defects associated with PID are currently available for the systemic immunodeficiency screening in newborns [15- 18]. TRECs and KRECs are formed during the genetic rearrangement necessary for the adequate T- and B-lymphocyte maturation. These DNA circles exhibit a dilution pattern, as they are not replicated during mitosis. Thus, newborns with severe PIDs associated with T- and/or B-cell lymphopenias, present low TRECs and KRECs copy numbers, regardless of the molecular aetiology of the underlying PID. This is important Page 7 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review rates of false-positive results but also in expanding knowledge regarding immune maturation. All external control samples from the CDC were correctly classified. Similarly, the seven samples from previously diagnosed patients with PID with T- and/or B-cell lymphopenia would have been picked up with the assay and the chosen cut-offs. The results from our center and other studies have proven the test to be highly reliable, relatively non-invasive and cost-effective making the nationwide implementation of screening for PID in Spain and other European countries attractive [9,30]. The purely technical costs of the in-house TRECs/KRECs/ACTB-assay used here are 1.46€/sample compared with 4.67€/sample of the commercial TREC assays used in the USA [9,24]. Low costs, the potential to detect and further characterize a broader spectrum of severe PID may favor the use of the TREC/KREC/ACTB-assay [1,7,9,17]. In summary, this study provides new data from the currently largest prospective cohort in Europe using the triplex TREC/KREC/ACTB-assay for NBS. It confirms the high diagnostic accuracy and robustness of the technique. TREC and KREC levels show a significant correlation and association between GA and BW in the Southern Spanish population. None of the preterm neonates showed isolated abnormal KRECs suggesting that the additional KREC determination would not increase re-punching and re-testing rates. The impact of immunosuppressive therapy during pregnancy on the results of KRECs remains to be established. Importantly, optimization of DBS preparation is needed in the future. Page 14 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review References 1. Borte S, Wang N, Oskarsdottir S, von Döbeln U, Hammarström L. Newborn screening for primary immunodeficiency: beyond SCID and XLA. Ann N Y Acad Sci 2011:1246:118-30. 2. Gathmann B, Binder N, Ehl S, Kindle G; ESID Registry Working Party. The European internet-based patient and research database for primary immunodeficiencies: update 2011. Clin Exp Immunol 2012:167:479-91. 3. Kwan A, Abraham RS, Currier R, Brower A, Andruszewski K, Abbott JK, et al. Newborn screening for combined immunodeficiency in 11 screening programs in the United States. JAMA 2014: 312:729-38. 4. Subbarayan A, Colarusso G, Hughes SM, Gennery AR, Slatter M, Cant AJ. Clinical features that identify children with primary immunodeficiency. Pediatrics 2011:127:810-816. 5. Brown L, Xu-Bayford J, Allwood Z, Slatter M, Cant A, Davies EG. Neonatal diagnosis of severe combined immunodeficiency leads to significantly improved survival outcome: the case of newborn screening. Blood 2011:17:2443-2446. 6. Chan A, Scalchunes C, Boyle M, Puck JM. Early vs. delayed diagnosis of severe combined immunodeficiency: a family perspective survey. Clin Immunol 2011: 138:3-8. 7. Borte S, von Döbeln U, Hammarström L. Guidelines for newborn screening of primary immunodeficiency diseases. Curr Opin Hematol. 2013:20:48-54. 8. Modell V, Knaus M, Modell F. An analysis and decision tool to measure cost benefit of newborn screening for severe combined immunodeficiency (SCID) and related T-cell lymphopenia. Immunol Res 2014:60:145-52. 9. Van der Spek J, Groenwold RH, van der Burg M, van Montfrans JM. TREC based Newborn Screening for Severe Combined Immunodeficiency Disease: A Systematic Review. J Clin Immunol 2015:35:416-30. 10. Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, et al. A modified γ-retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med 2014:371:1407-17. Page 15 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 11. Pai-SY, Logan BR, Griffith LM, Buckley RH, Parrott RE, Dvorak CC, et al. Transplantation outcome for severe combined immunodeficiency, 2000-2009. N Engl J Med 2014:371:434-46. 12. Bakare N, Menschik D, Tiernan R, Hua W, Martin D. Severe combined immunodeficiency (SCID) and rotavirus vaccination: reports to the Vaccine Adverse Events Reporting System (VAERS). Vaccine 2010:28:6609-12. 13. Marciano BE, Huang CY, Joshi G, Rezaei N, Carvalho BC, Allwood Z,et al. BCG vaccination in patients with severe combined immunodeficiency: complications, risks, and vaccination policies. J Allergy Clin Immunol. 2014 Apr;133(4):1134-41 14. Plebani A, Soresina A, Rondelli R, Amato GM, Azzari C, Cardinale F, et al. Clinical, immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study. Clin Immunol. 2002;104:221-30 15. Morinishi Y, Imai K, Nakagawa N, Sato H, Horiuchi K, Ohtsuka Y, et al. Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal guthrie cards. J Pediatr. 2009:155:829-33. 16. van Zelm MC, van der Burg M, Langerak AW, van Dongen JJ. PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders. Front Immunol 2011:4:2:12. 17. Borte S, von Döbeln U, Fasth A, Wang N, Janzi M, Winiarski J, et al. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR. Blood. 2012:119:2552-5. 18. Ombrone D, Giocaliere E, Forni G, Malvagia S, la Marca G. Expanded newborn screening by mass spectrometry: New test, future perspectives. Mass Spectrom Rev. 2015. doi: 10.1002/mas.21463. 19. Chinen J, Notarangelo LD, Shearer WT. Advances in basic and clinical immunology in 2014. J Allergy Clin Immunol. 2015:135:1132-1141. 20. http://primaryimmune.org/idf-advocacy-center/idf-scid-newborn-screening- campaign/ Page 16 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 21. Audrain M, Thomas C, Mirallie S, Bourgeois N, Sebille V, Rabetrano H, et al. Evaluation of the T-cell receptor excision circle assay performances for severe combined immunodeficiency neonatal screening on Guthrie cards in a French single centre study. Clin Immunol. 2014:150:137-9. 22. Adams SP, Rashid S, Premachandra T, Harvey K, Ifederu A, Wilson MC, et al. Screening of neonatal UK dried blood spots using a duplex TREC screening assay. J Clin Immunol. 2014:34:323-30. 23. Olbrich P, de Felipe B, Delgado-Pecellin C, Rodero R, Rojas P, Aguayo J, et al. [A first pilot study on the neonatal screening of primary immunodeficiencies in Spain: TRECS and KRECS identify severe T- and B-cell lymphopenia]. An Pediatr (Barc). 2014:81:310-7. 24. la Marca G, Canessa C, Giocaliere E, Romano F, Duse M, Malvagia S, et al. Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency. J Allergy Clin Immunol. 2013:131:1604-10. 25. Speckmann C, Neumann C, Borte S, la Marca G, Sass JO, Wiech E, et al. Delayed-onset adenosine deaminase deficiency: strategies for an early diagnosis. J Allergy Clin Immunol. 2012:130:991-4. 26. Kwan A, Puck JM. History and current status of newborn screening for severe combined immunodeficiency. Semin Perinatol. 2015:39:194-205. 27. Somech R, Lev A, Simon AJ, Korn D, Garty BZ, Amariglio N, et al. Newborn screening for severe T and B cell immunodeficiency in Israel: a pilot study. Isr Med Assoc 2013:15:404-9. 28. Vogel BH, Bonagura V, Weinberg GA, Ballow M, Isabelle J, DiAntonio L, et al. Newborn screening for SCID in New York State: experience from the first two years. J Clin Immunol 2014:34:289-303. 29. Rechavi E, Lev A, Lee YN, Simon AJ, Yinon Y, Lipitz S, et al. Timely and spatially regulated maturation of B and T cell repertoire during human fetal development. Sci Transl Med. 2015:7:276ra25. 30. Clément MC, Mahlaoui N, Mignot C, Le Bihan C, Rabetrano H, Hoang L, et al. Systemic neonatal screening for severe combined immunodeficiency and Page 17 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review severe T-cell lymphopenia: Analysis of cost-effectiveness based on French real field data. J Allergy Clin Immunol 2015:135:1589-93. Page 18 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 4 TRECS (copies/punch) 6 K RECS (copies/punch ) Page 19 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Table 1: Results of TREC/KREC/ACTB PCR-assay of DBS in relation to gestational age variables and birth weight p values were calculated comparing gestational age and birth weight of preterms using corresponding term neonates as the reference category; NA not applicable Gestational age N (%) TRECs/punch p KRECs/punch P ACTB/punch ≤28 38 0.7 36 [17-88] <0,001 32 [22-60] 0,02 1714 [1093-2521] 29-31 43 0.8 59 [40-81] <0,001 33 [18-54] 0,003 1345 [888-1541] 32-36 459 8.0 85 [49-131] <0,001 37 [20-64] <0,001 1498 [1001-2230] ≥37 3848 74.6 96 [62-141] 1 46 [27-75] 1 1673 [1117-2440] unknown 772 15 92 [61-136] NA 47 [26-76] NA 1649 [1088-2535] Birth weight N (%) TRECs/punch p KRECs/punch ACTB/punch <1000 28 0.5 30 [14-45] <0,001 32[15-41] 0,003 1512[900-2118] 1000-1499 64 1.2 75 [39-107] <0,001 32 [20-55] 0,001 1434 [1140-2222] 1500-2499 496 9.6 87 [54-125] <0,001 37 [21-60] <0,001 1526 [1032-2211] ≥2500 4092 79.3 95 [62-140] 1 46 [27-75] 1 1663 [1102-2441] unknown 480 9.3 96 [62-148] NA 49 [25-80] NA 1728 [1142-2604] TOTAL (5160) 5160 100 93 [60-139] 45 [26-74] 1649 [1100-2430] Page 20 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Table 2a) Summary of false positive results from prospective DBS Patient GA/GENDER 1 st TREC (copies/ punch ) 1 st KREC (copies/ punch ) 1 st ACTB (copies/ punch ) 2 nd TREC * (copies/ punch ) 2 nd KREC * (copies/ punch ) 2 nd ACTB * (copies/ punch ) 1 st LSS** (cells/µl) 2 nd LSS (cells/µl) Observations Diagnosis P1 32/female 1 4 1852 NA NA NA Total 500 CD3+ NA (%) CD19+ NA (%) NA Died age 1 month Trisomy, Monosomy (46,XX,der(7)t(7;15)(p2 2;q22)mat), Hydrops fetalis, Ebstein anomaly P2 25/male 0 0 4 4 8 7199 Total 2184 CD3+ 1366 (63%) CD19+ 367 (17%) NA Extreme preterm Blood product dependent P3 26/male 1 20 356 0 27 2592 Total 2424 CD3+ 1484 (60%) CD19+ 498 (20%) Total 7493 CD3+ 2454 (35%) CD19+ 4500 (57%) Extreme preterm Blood product dependent TOF IVIG dependent FU P4 Term/female 92 1 2953 NA NA NA NA NA Azathioprine during pregnancy* P5 Term/male 154 3 3589 61 1 863 Total 1700 CD3+ NA (%) CD19+ NA (%) NA Azathioprine during pregnancy** Page 21 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review * Mother diagnosed with Crohn´s disease; ** Mother diagnosed with SLE; * 2 nd sample was taken at 2 weeks of life, **1 st LSS were determined within 2 weeks from time of DBS result FU follow-up; GA gestational age; IVIG intravenous immunoglobulin; LSS lymphocyte subsets; NA not available or refused by guardians; TOF Tetralogy of Fallot Page 22 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review Table 2b) Summary results from internal true positive controls Control AGE at Time of DBS (months) Gender TREC (copies/ punch) KREC (copies/ punch) ACTB (copies/ punch) LSS (cells/µl) 1 st available sample Diagnosis C1 9.6 Male 0 56 705 Total 611 CD3+ 30 (4,7%) CD19+ 428 (67,1%) T-B+SCID (undefined) C2 0.2 Male 0.5 0 1257 Total 11800 CD3+ 9440 (80%) CD19+ (<1%) T-B-SCID (RAG) C3 0.2 Female 4 0 722 Total 300 CD3+ 79 (45%) CD19+ 1 (2%) T-B-SCID (ADA) C4 0.2 Male 1.9 0.6 1182 Total 1740 CD3+ 1087 (78%) CD19+ 1 (0,1%) T low B-SCID (Omenn) C5 93 Male 23 0 3229 Total 3400 CD3+ 2598 (76%) CD19+ 2 (0,05%) XLA C6 60 Male 42 0 1980 Total 5594 CD3+ 5346 (92,9%) CD19+ 4 (0,1%) XLA C7 20 Male 0 7 805 Total 1678 CD3+ 664 (42,9%) CD19+ 314 (20,2%) Ataxia telangiectasia Page 23 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 7 pattern, as they are not replicated during mitosis. Thus, newborns with severe PIDs 108 associated with T- and/or B-cell lymphopenias, present low TRECs and KRECs copy 109 numbers, regardless of the molecular aetiology of the underlying PID. This is important 110 given their broad genetic heterogeneity [16-20]. 111 In the USA 32 states have implemented the systematic NBS for T-cell deficiencies using 112 real-time PCR quantifying TRECs in DBS [21]. In Europe, research groups in France and 113 the United Kingdom have tested this TREC based methodology in retrospective studies 114 [22,23]. More recently, a multiplex-PCR has been developed to detect not only T-cell 115 lymphopenias (e.g. SCID, through low levels of TRECs) but also B-cell lymphopenias 116 (through low levels of KRECs) that are associated with other severe PIDs such as XLA or 117 ataxia telangiectasia (AT). This technique was initially evaluated in a cohort of 2560 118 anonymized DBS in Sweden [18] and afterwards prospectively tested by our group in 119 Seville [24]. 120 Currently, no consensus exists regarding the optimal screening technique. Whilst TREC 121 based methods have been broadly used in the USA [3], there are reports about missed 122 SCID, which would have been identified with the TRECs/KRECs/ACTB multiplex-PCR or 123 tandem mass spectrometry [25-26]. KRECs measurement increases the diagnostic 124 accuracy for specific SCID phenotypes such as ADA-SCID and enables to identify other 125 PID such as AT, Nijmegen-Breakage-Syndrome and inherited agammaglobulinemias 126 [1,10,18,24]. However, some experts suggest, that the inclusion of KRECs may 127 substantially increase the rate of false-positive results, leading to an unacceptable 128 number of sample re-testing, creating unnecessary family anxiety, harm to healthy 129 newborns and increased costs especially in the preterm neonate group [27]. 130 In this article we present our experience with the TRECs/KRECs/ACTB-assay in the 1 st 131 year since its implementation providing data on the feasibility and performance and 132 describe the incidence and aetiology of T- and B-cell lymphopenias. 133 134 Material and Methods 135 136 Study design, setting and population 137 Page 30 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 8 On-going prospective, observational and longitudinal study to determine TRECs and 138 KRECs levels in DBS, obtained from neonates born in three hospitals in Seville, Spain 139 (annexe), between February 2014 and March 2015. The local ethics committees 140 approved this study. Samples were included once the legal guardians signed informed 141 consent forms. 142 Sample and data collection 143 Heel prick blood samples were dropped on Schleicher&Schuell filter paper between 144 the 3 rd and 5 th day post-partum as part of the routine neonatal screening process. In 145 case of prematurity (gestational age (GA) <37 weeks), heel pricks were repeated every 146 two weeks until week 37 of corrected GA, birth weight (BW) ≥2500g or normality in 147 the assay. Two 3.2mm discs were punched for each sample, and stored at 4°C until 148 use. Demographic and clinical data including gender, GA, BW was collected. 149 Sample processing and multiplex real-time PCR (TRECs/KRECs/ACTB-assay) 150 Samples were processed and analysed as previously described [24]. Briefly, after DNA 151 extraction from DBS samples and DNA purification (DNA Elution and Purification 152 Solution, Qiagen, Maryland, USA) TRECs, KRECs, and β-actin (ACTB) copy numbers 153 were determined. The amount of ACTB copy numbers indicates the efficacy of DNA 154 extraction from DBS samples. 155 156 Quality controls 157 Internal controls (designed and obtained by plasmid cloning [18]) and 20 blinded 158 external controls provided by the NBS Quality Assurance Program of the Centers for 159 Disease Control and Prevention (CDC), USA were analysed. In addition, seven control 160 samples obtained from previously diagnosed patients from four Spanish 161 immunodeficiency units were included. 162 163 Definition and interpretation of results 164 Based on our previous experience the following cut-off scores for an estimated 99.8% 165 sensitivity in detecting severe T- and/or B-cell lymphopenias were defined as 166 following: TRECs<6/punch, KRECs<4/punch, ACTB>700/punch [22]. False-positive 167 Page 31 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 9 results were defined as values below the established cut-offs for TRECs or KRECs in 168 absence of SCID or inherited agammaglobulinemia, respectively. In case of abnormal 169 or inconclusive results, a new punch from the same Guthrie card was obtained and the 170 PCR-assay repeated (re-test). Only pathologic results in the re-test led to the request of 171 a new heel prick sample (re-call) and the confirmation of a result below the 172 established cut-off was followed by an assessment of the newborn. In case of 173 insufficient material of the 1 st Guthrie card despite re-punching, re-call with extraction 174 of a 2 nd DBS was offered, but not routinely performed. 175 176 Data analysis 177 Qualitative variables are shown as absolute frequencies and percentages. Quantitative 178 variables were tested (Kolmogorov–Smirnov test, or Shapiro–Wilk test) for normal 179 distribution and are expressed as mean±standard deviation (SD) or as median and 180 interquartile range (IQR), when appropriate. Rates for abnormal, inconclusive, and 181 normal results in the TRECs/KRECs-assay were calculated (CI 95%). To assess the assay 182 reliability, the proportion of false-positive results was calculated. For qualitative 183 variables the chi-squared or the Fisher´s exact test were applied to estimate variable 184 associations with T- and B-cell lymphopenias. For quantitative variables the Student's t-185 test for independent samples or the Mann–Whitney U test were performed. 186 Correlations were assessed by means of the Pearson tests. p-values of <0,05 were 187 considered as statistical significant. All statistical operations were performed using the 188 IBM software SPSS Statistics version 20. 189 190 Results 191 Demographic and clinical data of the cohort 192 A total of 5279 samples were prospectively collected from 5160 neonates. 51.2% were 193 males; 74.6% were born at term (GA ≥37 weeks) and 79.3% had a BW ≥2500g. The 194 mean GA was 38.6 weeks (±2.3 weeks) and the mean BW was 3147g (±610g). 195 Information regarding BW or GA was incomplete for 480 (9.3%) and 772 (15.0%) valid 196 samples, respectively. 197 198 Page 32 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 10 Sample quality and assay performance 199 Out of 5160 neonates, 109 showed low copies of ACTB; 32 of which could not be 200 repeated due to insufficient material (0.6%). Re-punch of the remaining 77 samples 201 (1.5%) revealed 10 positive results and were re-called (0.19%); five had subsequently 202 normal results and the remaining five samples were confirmed to be positive (0.1%). 203 204 Influence of GA and BW on TRECs, KRECs and ACTB levels 205 Results of the TRECs/KRECs-assay, calculated for the subgroups of GA and BW, are 206 shown in Table 1. TRECs and KRECs copy numbers were significantly lower in preterm 207 neonates compared to term neonates (p<0.001). Similarly, newborns with a BW 208 <2500g showed lower TRECs and KRECs copy numbers than those with a BW ≥2500g 209 (p<0.001). A positive correlation between GA and BW with TRECs (r=0.117, p<0.001; 210 r=0.118, p<0.001) as well as with KRECs (r=0.05, p=0.001; r=0.052, p=0.001) was 211 observed. For both variables this association was more pronounced for the TRECs. No 212 differences were found when analysing the effect of GA and BW on the ACTB copy 213 numbers. 214 215 Positive results from prospectively tested DBS 216 As shown in Figure 1 and Table 2a five of the 5160 prospectively tested newborns 217 showed positive results in the assay. Patient 1 (P1) was a preterm female diagnosed 218 with a complex chromosomopathy. Confirmation of the initially positive results with a 219 second sample or lymphocyte subsets (LSS) determination could not be performed due 220 to the rapidly fatal course of this patient. Two other individuals (P2, P3) with positive 221 results in the 1 st test run were identified as blood product dependent extreme preterm 222 neonates. Low ACTB and TRECs were found in both individuals. In addition P3 also 223 showed reduced KREC copy numbers. All three patients had a low for age lymphocyte 224 count whilst LSS distribution of patients 2 and 3 was normal. A 2 nd sample of the later 225 demonstrated normal KRECs and ACTB levels whilst TRECs remained reduced. 226 Repeated LSS were subsequently normal for age in P3 but not available for P2. Both 227 patients are currently under follow-up. Low KRECs were detected in P4 and P5. A 228 telephone interview with the family revealed both mothers receiving 229 immunosuppressive treatment for systemic lupus erythematosus (SLE) and Crohn’s 230 Page 33 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 11 disease including azathioprine during pregnancy. Whereas the family of P4 refused to 231 perform a second heel prick, P5 demonstrated persistent low KREC levels at one 232 month of age. However, a 3 rd blood sample taken from P5 four weeks after stopping 233 breast-feeding showed a normal LSS and KREC levels (Table 2b). A simultaneous blood 234 sample taken from the mother of P5 revealed B-cell lymphopenia (39 cells/µl) and 235 pathological TRECs and KRECs copy numbers (0 and 1, respectively) whilst receiving 236 azathioprine treatment. 237 238 External control samples of the CDC 239 Four sets of blinded samples (n=20) were provided for quality assessment from the 240 CDC. All positive (low TREC copy numbers, β-actin above cut-off), and negative samples 241 (TREC and β-actin above cut-off) were correctly identified as shown in Figure 1, as it 242 was the case with DNA free samples (undetectable TREC/KREC/β-actin; not included in 243 Figure 1). 244 245 Samples from patients with known PID 246 Seven samples from patients with known underlying PID were included (Table 2b). 247 Although in all cases the first available sample was used for analysis, age variation was 248 broad (0.2-93 months). Four patients suffered from SCID (age range 0.2-9.6 months). 249 TREC levels in these patients were all below the cut-off (range 0-4 copies/µl). Similarly, 250 KRECs were abnormal in two subjects with a T-B-SCID phenotype. Two patients with 251 XLA were included, aged 60 and 93 months at first DBS, and KRECs were correctly 252 absent. One patient with AT had absent TRECs and KRECs in the lower limit. All 253 included samples showed normal ACTB levels. 254 255 Discussion 256 This is currently the largest prospective study in Europe using the triplex 257 TRECs/KRECs/ACTB-assay for NBS of severe T- and B-cell lymphopenias. Previous 258 studies with mainly retrospective design from Sweden, UK, France and Israel highlight 259 the importance for its implementation in Europe [18,22,23,28]. The USA data showed 260 the efficacy, necessity and cost-effectiveness of NBS for T-cell deficiencies [3,9,27]. No 261 consensus exists regarding the best screening methodology. Specifically the addition of 262 Page 34 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 12 KRECs to the assay is still under debate [1,10,27]. Algorithms for structured sample 263 processing, result interpretation and decisions regarding the clinical follow-up are 264 unanswered but important issues for all screening centers [10,27]. 265 In this ongoing study we prospectively collected and analyzed 5279 samples from 5160 266 newborns. Values of TRECs and KRECs were found to be similar to other previously 267 studied populations [18]. The initially chosen cut-off levels for TRECs and KRECs were 268 progressively optimized in order to avoid unnecessary sample drawing and analysis 269 related with unjustified harm to the newborn and additional costs [24]. Despite this 270 adjustment the re-punch rate remained high, being 1.5% whilst re-call rate was low 271 being 0.19%, complying with previous studies (10). Poor pre-analytic sample quality, 272 mainly due to inappropriately prepared DBS (insufficient blood quantity and storage) 273 resulted in low ACTB and was the most frequently identified reason for re-punching. 274 Implementation of training programs and optimized communication with primary care 275 centers nursery are needed to improve routine NBS performance and subsequently re-276 punch and re-call rates. The close collaboration with the regional screening center 277 enabled us to avoid additional harm to newborns with insufficient DBS. Only two 278 newborns were re-called, the remaining 8 newborns being previously contacted by the 279 metabolic screening center for routine NBS repetition. We found an association 280 between TRECs and KRECs levels with the demographic variables GA and BW. Lower 281 TREC values have been previously described in preterm neonates [3,23,29], whereas 282 no reports exist on the influence of GA and BW on the KREC levels. Only two extreme 283 preterm neonates admitted to the neonatal intensive care unit showed TREC levels 284 below the established cut-off whilst none had pathological KREC levels, suggesting the 285 addition of KRECs to the assay not to increase the number of false-positive results in 286 the preterm population. Overall the impact of GA and BW on TRECs was stronger 287 compared to the KRECs. This is in line with a recent study investigating the maturation 288 of the human immune system at different gestational ages, demonstrating B-cell 289 development to precede T-cell maturation in human fetuses [30]. 290 Two term neonates had KRECs below the cut-off with normal TREC values. Both 291 neonates were born to mothers receiving immunosuppressive therapy during 292 pregnancy with azathioprine for SLE and Crohn’s disease, highlighting the importance 293 of a complete clinical history as part of the second tier testing. The potential 294 Page 35 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 13 association of immunosuppressive therapy and abnormal KRECs but normal TRECs has 295 not been previously described and may be important, not only in light of incremented 296 rates of false-positive results but also in expanding knowledge regarding immune 297 maturation. Noteworthy, a repeat lymphocyte LSS as well as the determination of 298 KRECS copies showed subsequently normal results of patient 5 (P5). Importantly, P5 299 received breast milk when the 2 nd blood sample was taken and this was associated 300 with abnormal low KRECs copies. In contrast, KRECs copies were normal four weeks 301 after stopping breast-feeding as was a repeat LSS (3 rd blood sample), whilst the mother 302 continued on azathioprine maintaining abnormal low KRECs copies and B-cell 303 lymphopenia, suggesting a transitional toxic effect of azathioprine on KRECs. This 304 observation might be associated with potential “false” low KRECs levels and 305 unnecessary repeat blood sampling. 306 All external control samples from the CDC were correctly classified. Similarly, the seven 307 DBS from previously diagnosed patients with PID with T- and/or B-cell lymphopenia 308 would have been picked up with the assay and the chosen cut-offs, thereby supporting 309 previous studies, who have shown the test to be reliable, relatively non-invasive and 310 cost-effective making the nationwide implementation of screening for PID in Spain and 311 other European countries attractive [10,31]. The purely technical costs of the in-house 312 TRECs/KRECs/ACTB-assay used here are 1.76€/sample compared with 4.67€/sample of 313 the commercial TREC assays used in the USA [9,24]. Low costs, the potential to detect 314 and further characterize a broader spectrum of severe PID may favor the use of the 315 TREC/KREC/ACTB-assay [1,8,10,18]. 316 317 In summary, this study provides new data from the currently largest prospective 318 cohort in Europe using the triplex TREC/KREC/ACTB-assay for NBS. It confirms the high 319 diagnostic accuracy and robustness of the technique. TREC and KREC levels show a 320 significant correlation and association between GA and BW in the Southern Spanish 321 population. None of the preterm neonates showed isolated abnormal KRECs 322 suggesting that the additional KREC determination would not increase re-punching and 323 re-testing rates. The impact of immunosuppressive therapy during pregnancy on the 324 results of KRECs remains to be established. Importantly, optimization of DBS 325 preparation is needed in the future. 326 Page 36 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 14 References 327 328 1. Borte S, Wang N, Oskarsdottir S, von Döbeln U, Hammarström L. Newborn 329 screening for primary immunodeficiency: beyond SCID and XLA. Ann N Y Acad 330 Sci 2011:1246:118-30. 331 2. Gathmann B, Binder N, Ehl S, Kindle G; ESID Registry Working Party. The 332 European internet-based patient and research database for primary 333 immunodeficiencies: update 2011. Clin Exp Immunol 2012:167:479-91. 334 3. Kwan A, Abraham RS, Currier R, Brower A, Andruszewski K, Abbott JK, et al. 335 Newborn screening for combined immunodeficiency in 11 screening programs 336 in the United States. JAMA 2014: 312:729-38. 337 4. Winkelstein JA, Marino MC, Lederman HM, Jones SM, Sullivan K, Burks AW, et 338 al. X-linked agammaglobulinemia: report on a United States registry of 201 339 patients. Medicine . 2006;85(4):193-202. 340 5. Subbarayan A, Colarusso G, Hughes SM, Gennery AR, Slatter M, Cant AJ. Clinical 341 features that identify children with primary immunodeficiency. Pediatrics 342 2011:127:810-816. 343 6. Brown L, Xu-Bayford J, Allwood Z, Slatter M, Cant A, Davies EG. Neonatal 344 diagnosis of severe combined immunodeficiency leads to significantly improved 345 survival outcome: the case of newborn screening. Blood 2011:17:2443-2446. 346 7. Chan A, Scalchunes C, Boyle M, Puck JM. Early vs. delayed diagnosis of severe 347 combined immunodeficiency: a family perspective survey. Clin Immunol 2011: 348 138:3-8. 349 8. Borte S, von Döbeln U, Hammarström L. Guidelines for newborn screening of 350 primary immunodeficiency diseases. Curr Opin Hematol. 2013:20:48-54. 351 9. Modell V, Knaus M, Modell F. An analysis and decision tool to measure cost 352 benefit of newborn screening for severe combined immunodeficiency (SCID) 353 and related T-cell lymphopenia. Immunol Res 2014:60:145-52. 354 10. Van der Spek J, Groenwold RH, van der Burg M, van Montfrans JM. TREC based 355 Newborn Screening for Severe Combined Immunodeficiency Disease: A 356 Systematic Review. J Clin Immunol 2015:35:416-30. 357 Page 37 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 15 11. Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, et al. A 358 modified γ-retrovirus vector for X-linked severe combined immunodeficiency. N 359 Engl J Med 2014:371:1407-17. 360 12. Pai-SY, Logan BR, Griffith LM, Buckley RH, Parrott RE, Dvorak CC, et al. 361 Transplantation outcome for severe combined immunodeficiency, 2000-2009. 362 N Engl J Med 2014:371:434-46. 363 13. Bakare N, Menschik D, Tiernan R, Hua W, Martin D. Severe combined 364 immunodeficiency (SCID) and rotavirus vaccination: reports to the Vaccine 365 Adverse Events Reporting System (VAERS). Vaccine 2010:28:6609-12. 366 14. Marciano BE, Huang CY, Joshi G, Rezaei N, Carvalho BC, Allwood Z,et al. BCG 367 vaccination in patients with severe combined immunodeficiency: 368 complications, risks, and vaccination policies. J Allergy Clin Immunol. 2014 369 Apr;133(4):1134-41 370 15. Plebani A, Soresina A, Rondelli R, Amato GM, Azzari C, Cardinale F, et al. 371 Clinical, immunological, and molecular analysis in a large cohort of patients 372 with X-linked agammaglobulinemia: an Italian multicenter study. Clin Immunol. 373 2002;104:221-30 374 16. Morinishi Y, Imai K, Nakagawa N, Sato H, Horiuchi K, Ohtsuka Y, et al. 375 Identification of severe combined immunodeficiency by T-cell receptor excision 376 circles quantification using neonatal guthrie cards. J Pediatr. 2009:155:829-33. 377 17. van Zelm MC, van der Burg M, Langerak AW, van Dongen JJ. PID comes full 378 circle: applications of V(D)J recombination excision circles in research, 379 diagnostics and newborn screening of primary immunodeficiency disorders. 380 Front Immunol 2011:4:2:12. 381 18. Borte S, von Döbeln U, Fasth A, Wang N, Janzi M, Winiarski J, et al. Neonatal 382 screening for severe primary immunodeficiency diseases using high-throughput 383 triplex real-time PCR. Blood. 2012:119:2552-5. 384 19. Ombrone D, Giocaliere E, Forni G, Malvagia S, la Marca G. Expanded newborn 385 screening by mass spectrometry: New test, future perspectives. Mass Spectrom 386 Rev. 2015. doi: 10.1002/mas.21463. 387 20. Chinen J, Notarangelo LD, Shearer WT. Advances in basic and clinical 388 immunology in 2014. J Allergy Clin Immunol. 2015:135:1132-1141. 389 Page 38 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 16 21. http://primaryimmune.org/idf-advocacy-center/idf-scid-newborn-screening-390 campaign/ 391 22. Audrain M, Thomas C, Mirallie S, Bourgeois N, Sebille V, Rabetrano H, et al. 392 Evaluation of the T-cell receptor excision circle assay performances for severe 393 combined immunodeficiency neonatal screening on Guthrie cards in a French 394 single centre study. Clin Immunol. 2014:150:137-9. 395 23. Adams SP, Rashid S, Premachandra T, Harvey K, Ifederu A, Wilson MC, et al. 396 Screening of neonatal UK dried blood spots using a duplex TREC screening 397 assay. J Clin Immunol. 2014:34:323-30. 398 24. Olbrich P, de Felipe B, Delgado-Pecellin C, Rodero R, Rojas P, Aguayo J, et al. [A 399 first pilot study on the neonatal screening of primary immunodeficiencies in 400 Spain: TRECS and KRECS identify severe T- and B-cell lymphopenia]. An Pediatr 401 (Barc). 2014:81:310-7. 402 25. la Marca G, Canessa C, Giocaliere E, Romano F, Duse M, Malvagia S, et al. 403 Tandem mass spectrometry, but not T-cell receptor excision circle analysis, 404 identifies newborns with late-onset adenosine deaminase deficiency. J Allergy 405 Clin Immunol. 2013:131:1604-10. 406 26. Speckmann C, Neumann C, Borte S, la Marca G, Sass JO, Wiech E, et al. 407 Delayed-onset adenosine deaminase deficiency: strategies for an early 408 diagnosis. J Allergy Clin Immunol. 2012:130:991-4. 409 27. Kwan A, Puck JM. History and current status of newborn screening for severe 410 combined immunodeficiency. Semin Perinatol. 2015:39:194-205. 411 28. Somech R, Lev A, Simon AJ, Korn D, Garty BZ, Amariglio N, et al. Newborn 412 screening for severe T and B cell immunodeficiency in Israel: a pilot study. Isr 413 Med Assoc 2013:15:404-9. 414 29. Vogel BH, Bonagura V, Weinberg GA, Ballow M, Isabelle J, DiAntonio L, et al. 415 Newborn screening for SCID in New York State: experience from the first two 416 years. J Clin Immunol 2014:34:289-303. 417 30. Rechavi E, Lev A, Lee YN, Simon AJ, Yinon Y, Lipitz S, et al. Timely and spatially 418 regulated maturation of B and T cell repertoire during human fetal 419 development. Sci Transl Med. 2015:7:276ra25. 420 Page 39 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 6 Introduction 79 Severe T- and/or B-cell lymphopenias in the first days of life may indicate congenital 80 immune defects such as X-linked agammaglobulinemia (XLA) and severe combined 81 immunodeficiency (SCID) [1]. Whilst the incidence of SCID is considered to be at least 82 1:35.000 births in some US states, the true incidence in Spain is unknown. Although 83 SCID and XLA belong to the group of rare diseases, their incidences of 1:35,000 to 84 1:200,000 live births is still considerably high given their severity, particularly in SCID 85 patients [2,3,4]. The common lack of a suggestive family history and the absence of 86 specific clinical symptoms in the first weeks of life often lead to a delayed diagnosis, 87 associated with prolonged intensive care treatment, high morbidity and mortality [5-88 7]. 89 To date, no European country performs newborn screening (NBS) for primary 90 immunodeficiencies (PIDs) routinely, despite meeting most of the established 91 screening criteria, including the existence of relatively non-invasive screening 92 methodology, proven benefit of early diagnosis, presence of an initial asymptomatic 93 period, high morbidity and mortality, availability of curative or effective therapeutic 94 options and a favourable cost-benefit analysis [8-10]. 95 In contrast to most of the diseases included in the routine NBS, curative treatment 96 approaches for SCID patients exist in form of hematopoietic stem cell transplantation 97 (HSCT) or gene therapy [11,12]. Furthermore, early diagnosis avoids preventable 98 iatrogenic events related to live vaccines such as rotavirus [13,14]. In patients with 99 congenital agammaglobulinemias, prompt diagnosis and immunoglobulin substitution 100 have shown to improve overall outcomes [15]. 101 The quantification of two circular DNA fragments (T-cell-receptor-excision circles, 102 TRECs and kappa-deleting-recombination-excision circles, KRECs) in dried blood spots 103 (DBS) and tandem mass spectrometry for enzyme defects associated with PID are 104 currently available for the systemic immunodeficiency screening in newborns [16-19]. 105 TRECs and KRECs are formed during the genetic rearrangement necessary for the 106 adequate T- and B-lymphocyte maturation. These DNA circles exhibit a dilution 107 Page 46 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 7 pattern, as they are not replicated during mitosis. Thus, newborns with severe PIDs 108 associated with T- and/or B-cell lymphopenias, present low TRECs and KRECs copy 109 numbers, regardless of the molecular aetiology of the underlying PID. This is important 110 given their broad genetic heterogeneity [16-20]. 111 In the USA 32 states have implemented the systematic NBS for T-cell deficiencies using 112 real-time PCR quantifying TRECs in DBS [21]. In Europe, research groups in France and 113 the United Kingdom have tested this TREC based methodology in retrospective studies 114 [22,23]. More recently, a multiplex-PCR has been developed to detect not only T-cell 115 lymphopenias (e.g. SCID, through low levels of TRECs) but also B-cell lymphopenias 116 (through low levels of KRECs) that are associated with other severe PIDs such as XLA or 117 ataxia telangiectasia (AT). This technique was initially evaluated in a cohort of 2560 118 anonymized DBS in Sweden [18] and afterwards prospectively tested by our group in 119 Seville [24]. 120 Currently, no consensus exists regarding the optimal screening technique. Whilst TREC 121 based methods have been broadly used in the USA [3], there are reports about missed 122 SCID, which would have been identified with the TRECs/KRECs/ACTB multiplex-PCR or 123 tandem mass spectrometry [25-26]. KRECs measurement increases the diagnostic 124 accuracy for specific SCID phenotypes such as ADA-SCID and enables to identify other 125 PID such as AT, Nijmegen-Breakage-Syndrome and inherited agammaglobulinemias 126 [1,10,18,24]. However, some experts suggest, that the inclusion of KRECs may 127 substantially increase the rate of false-positive results, leading to an unacceptable 128 number of sample re-testing, creating unnecessary family anxiety, harm to healthy 129 newborns and increased costs especially in the preterm neonate group [27]. 130 In this article we present our experience with the TRECs/KRECs/ACTB-assay in the 1 st 131 year since its implementation providing data on the feasibility and performance and 132 describe the incidence and aetiology of T- and B-cell lymphopenias. 133 134 Material and Methods 135 136 Study design, setting and population 137 Page 47 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 8 On-going prospective, observational and longitudinal study to determine TRECs and 138 KRECs levels in DBS, obtained from neonates born in three hospitals in Seville, Spain 139 (annexe), between February 2014 and March 2015. The local ethics committees 140 approved this study. Samples were included once the legal guardians signed informed 141 consent forms. 142 Sample and data collection 143 Heel prick blood samples were dropped on Schleicher&Schuell filter paper between 144 the 3 rd and 5 th day post-partum as part of the routine neonatal screening process. In 145 case of prematurity (gestational age (GA) <37 weeks), heel pricks were repeated every 146 two weeks until week 37 of corrected GA, birth weight (BW) ≥2500g or normality in 147 the assay. Two 3.2mm discs were punched for each sample, and stored at 4°C until 148 use. Demographic and clinical data including gender, GA, BW was collected. 149 Sample processing and multiplex real-time PCR (TRECs/KRECs/ACTB-assay) 150 Samples were processed and analysed as previously described [24]. Briefly, after DNA 151 extraction from DBS samples and DNA purification (DNA Elution and Purification 152 Solution, Qiagen, Maryland, USA) TRECs, KRECs, and β-actin (ACTB) copy numbers 153 were determined. The amount of ACTB copy numbers indicates the efficacy of DNA 154 extraction from DBS samples. 155 156 Quality controls 157 Internal controls (designed and obtained by plasmid cloning [18]) and 20 blinded 158 external controls provided by the NBS Quality Assurance Program of the Centers for 159 Disease Control and Prevention (CDC), USA were analysed. In addition, seven control 160 samples obtained from previously diagnosed patients from four Spanish 161 immunodeficiency units were included. 162 163 Definition and interpretation of results 164 Based on our previous experience the following cut-off scores for an estimated 99.8% 165 sensitivity in detecting severe T- and/or B-cell lymphopenias were defined as 166 following: TRECs<6/punch, KRECs<4/punch, ACTB>700/punch [22]. False-positive 167 Page 48 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 9 results were defined as values below the established cut-offs for TRECs or KRECs in 168 absence of SCID or inherited agammaglobulinemia, respectively. In case of abnormal 169 or inconclusive results, a new punch from the same Guthrie card was obtained and the 170 PCR-assay repeated (re-test). Only pathologic results in the re-test led to the request of 171 a new heel prick sample (re-call) and the confirmation of a result below the 172 established cut-off was followed by an assessment of the newborn. In case of 173 insufficient material of the 1 st Guthrie card despite re-punching, re-call with extraction 174 of a 2 nd DBS was offered, but not routinely performed. 175 176 Data analysis 177 Qualitative variables are shown as absolute frequencies and percentages. Quantitative 178 variables were tested (Kolmogorov–Smirnov test, or Shapiro–Wilk test) for normal 179 distribution and are expressed as mean±standard deviation (SD) or as median and 180 interquartile range (IQR), when appropriate. Rates for abnormal, inconclusive, and 181 normal results in the TRECs/KRECs-assay were calculated (CI 95%). To assess the assay 182 reliability, the proportion of false-positive results was calculated. For qualitative 183 variables the chi-squared or the Fisher´s exact test were applied to estimate variable 184 associations with T- and B-cell lymphopenias. For quantitative variables the Student's t-185 test for independent samples or the Mann–Whitney U test were performed. 186 Correlations were assessed by means of the Pearson tests. p-values of <0,05 were 187 considered as statistical significant. All statistical operations were performed using the 188 IBM software SPSS Statistics version 20. 189 190 Results 191 Demographic and clinical data of the cohort 192 A total of 5279 samples were prospectively collected from 5160 neonates. 51.2% were 193 males; 74.6% were born at term (GA ≥37 weeks) and 79.3% had a BW ≥2500g. The 194 mean GA was 38.6 weeks (±2.3 weeks) and the mean BW was 3147g (±610g). 195 Information regarding BW or GA was incomplete for 480 (9.3%) and 772 (15.0%) valid 196 samples, respectively. 197 198 Page 49 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 10 Sample quality and assay performance 199 Out of 5160 neonates, 109 showed low copies of ACTB; 32 of which could not be 200 repeated due to insufficient material (0.6%). Re-punch of the remaining 77 samples 201 (1.5%) revealed 10 positive results and were re-called (0.19%); five had subsequently 202 normal results and the remaining five samples were confirmed to be positive (0.1%). 203 204 Influence of GA and BW on TRECs, KRECs and ACTB levels 205 Results of the TRECs/KRECs-assay, calculated for the subgroups of GA and BW, are 206 shown in Table 1. TRECs and KRECs copy numbers were significantly lower in preterm 207 neonates compared to term neonates (p<0.001). Similarly, newborns with a BW 208 <2500g showed lower TRECs and KRECs copy numbers than those with a BW ≥2500g 209 (p<0.001). A positive correlation between GA and BW with TRECs (r=0.117, p<0.001; 210 r=0.118, p<0.001) as well as with KRECs (r=0.05, p=0.001; r=0.052, p=0.001) was 211 observed. For both variables this association was more pronounced for the TRECs. No 212 differences were found when analysing the effect of GA and BW on the ACTB copy 213 numbers. 214 215 Positive results from prospectively tested DBS 216 As shown in Figure 1 and Table 2a five of the 5160 prospectively tested newborns 217 showed positive results in the assay. Patient 1 (P1) was a preterm female diagnosed 218 with a complex chromosomopathy. Confirmation of the initially positive results with a 219 second sample or lymphocyte subsets (LSS) determination could not be performed due 220 to the rapidly fatal course of this patient. Two other individuals (P2, P3) with positive 221 results in the 1 st test run were identified as blood product dependent extreme preterm 222 neonates. Low ACTB and TRECs were found in both individuals. In addition P3 also 223 showed reduced KREC copy numbers. All three patients had a low for age lymphocyte 224 count whilst LSS distribution of patients 2 and 3 was normal. A 2 nd sample of the later 225 demonstrated normal KRECs and ACTB levels whilst TRECs remained reduced. 226 Repeated LSS were subsequently normal for age in P3 but not available for P2. Both 227 patients are currently under follow-up. Low KRECs were detected in P4 and P5. A 228 telephone interview with the family revealed both mothers receiving 229 immunosuppressive treatment for systemic lupus erythematosus (SLE) and Crohn’s 230 Page 50 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 11 disease including azathioprine during pregnancy. Whereas the family of P4 refused to 231 perform a second heel prick, P5 demonstrated persistent low KREC levels at one 232 month of age. However, a 3 rd blood sample taken from P5 four weeks after stopping 233 breast-feeding showed a normal LSS and KREC levels (Table 2b). A simultaneous blood 234 sample taken from the mother of P5 revealed B-cell lymphopenia (39 cells/µl) and 235 pathological TRECs and KRECs copy numbers (0 and 1, respectively) whilst receiving 236 azathioprine treatment. 237 238 External control samples of the CDC 239 Four sets of blinded samples (n=20) were provided for quality assessment from the 240 CDC. All positive (low TREC copy numbers, β-actin above cut-off), and negative samples 241 (TREC and β-actin above cut-off) were correctly identified as shown in Figure 1, as it 242 was the case with DNA free samples (undetectable TREC/KREC/β-actin; not included in 243 Figure 1). 244 245 Samples from patients with known PID 246 Seven samples from patients with known underlying PID were included (Table 2b). 247 Although in all cases the first available sample was used for analysis, age variation was 248 broad (0.2-93 months). Four patients suffered from SCID (age range 0.2-9.6 months). 249 TREC levels in these patients were all below the cut-off (range 0-4 copies/µl). Similarly, 250 KRECs were abnormal in two subjects with a T-B-SCID phenotype. Two patients with 251 XLA were included, aged 60 and 93 months at first DBS, and KRECs were correctly 252 absent. One patient with AT had absent TRECs and KRECs in the lower limit. All 253 included samples showed normal ACTB levels. 254 255 Discussion 256 This is currently the largest prospective study in Europe using the triplex 257 TRECs/KRECs/ACTB-assay for NBS of severe T- and B-cell lymphopenias. Previous 258 studies with mainly retrospective design from Sweden, UK, France and Israel highlight 259 the importance for its implementation in Europe [18,22,23,28]. The USA data showed 260 the efficacy, necessity and cost-effectiveness of NBS for T-cell deficiencies [3,9,27]. No 261 consensus exists regarding the best screening methodology. Specifically the addition of 262 Page 51 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 12 KRECs to the assay is still under debate [1,10,27]. Algorithms for structured sample 263 processing, result interpretation and decisions regarding the clinical follow-up are 264 unanswered but important issues for all screening centers [10,27]. 265 In this ongoing study we prospectively collected and analyzed 5279 samples from 5160 266 newborns. Values of TRECs and KRECs were found to be similar to other previously 267 studied populations [18]. The initially chosen cut-off levels for TRECs and KRECs were 268 progressively optimized in order to avoid unnecessary sample drawing and analysis 269 related with unjustified harm to the newborn and additional costs [24]. Despite this 270 adjustment the re-punch rate remained high, being 1.5% whilst re-call rate was low 271 being 0.19%, complying with previous studies (10). Poor pre-analytic sample quality, 272 mainly due to inappropriately prepared DBS (insufficient blood quantity and storage) 273 resulted in low ACTB and was the most frequently identified reason for re-punching. 274 Implementation of training programs and optimized communication with primary care 275 centers nursery are needed to improve routine NBS performance and subsequently re-276 punch and re-call rates. The close collaboration with the regional screening center 277 enabled us to avoid additional harm to newborns with insufficient DBS. Only two 278 newborns were re-called, the remaining 8 newborns being previously contacted by the 279 metabolic screening center for routine NBS repetition. We found an association 280 between TRECs and KRECs levels with the demographic variables GA and BW. Lower 281 TREC values have been previously described in preterm neonates [3,23,29], whereas 282 no reports exist on the influence of GA and BW on the KREC levels. Only two extreme 283 preterm neonates admitted to the neonatal intensive care unit showed TREC levels 284 below the established cut-off whilst none had pathological KREC levels, suggesting the 285 addition of KRECs to the assay not to increase the number of false-positive results in 286 the preterm population. Overall the impact of GA and BW on TRECs was stronger 287 compared to the KRECs. This is in line with a recent study investigating the maturation 288 of the human immune system at different gestational ages, demonstrating B-cell 289 development to precede T-cell maturation in human fetuses [30]. 290 Two term neonates had KRECs below the cut-off with normal TREC values. Both 291 neonates were born to mothers receiving immunosuppressive therapy during 292 pregnancy with azathioprine for SLE and Crohn’s disease, highlighting the importance 293 of a complete clinical history as part of the second tier testing. The potential 294 Page 52 of 59 Scholarone, Inc. Support (434) 964 4100 Pediatric Allergy and Immunology For Peer Review 13 association of immunosuppressive therapy and abnormal KRECs but normal TRECs has 295 not been previously described and may be important, not only in light of incremented 296 rates of false-positive results but also in expanding knowledge regarding immune 297 maturation. Noteworthy, a repeat lymphocyte LSS as well as the determination of 298 KRECS copies showed subsequently normal results of patient 5 (P5). Importantly, P5 299 received breast milk when the 2 nd blood sample was taken and this was associated 300 with abnormal low KRECs copies. In contrast, KRECs copies were normal four weeks 301 after stopping breast-feeding as was a repeat LSS (3 rd blood sample), whilst the mother 302 continued on azathioprine maintaining abnormal low KRECs copies and B-cell 303 lymphopenia, suggesting a transitional toxic effect of azathioprine on KRECs. This 304 observation might be associated with potential “false” low KRECs levels and 305 unnecessary repeat blood sampling. 306 All external control samples from the CDC were correctly classified. Similarly, the seven 307 DBS from previously diagnosed patients with PID with T- and/or B-cell lymphopenia 308 would have been picked up with the assay and the chosen cut-offs, thereby supporting 309 previous studies, who have shown the test to be reliable, relatively non-invasive and 310 cost-effective making the nationwide implementation of screening for PID in Spain and 311 other European countries attractive [10,31]. The purely technical costs of the in-house 312 TRECs/KRECs/ACTB-assay used here are 1.76€/sample compared with 4.67€/sample of 313 the commercial TREC assays used in the USA [9,24]. Low costs, the potential to detect 314 and further characterize a broader spectrum of severe PID may favor the use of the 315 TREC/KREC/ACTB-assay [1,8,10,18]. 316 317 In summary, this study provides new data from the currently largest prospective 318 cohort in Europe using the triplex TREC/KREC/ACTB-assay for NBS. It confirms the high 319 diagnostic accuracy and robustness of the technique. TREC and KREC levels show a 320 significant correlation and association between GA and BW in the Southern Spanish 321 population. None of the preterm neonates showed isolated abnormal KRECs 322 suggesting that the additional KREC determination would not increase re-punching and 323 re-testing rates. The impact of immunosuppressive therapy during pregnancy on the 324 results of KRECs remains to be established. Importantly, optimization of DBS 325 preparation is needed in the future. 326 Page 53 of 59 Scholarone, Inc. 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