P Olbrich1, B de Felipe1, C Delgado-Pecellin2, R Rodero3, P Rojas3, J Aguayo3, J Marquez4, J Casanovas4, B Sánchez5, J M Lucena5, P Ybot-Gonzalez6, S Borte7, O Neth8. 1. Sección de Infectología e Inmunodeficiencias, Unidad de Pediatría, Hospital Virgen del Rocío, Sevilla, Instituto de Biomedicina de Sevilla (IBiS), Sevilla, España. 2. Unidad de Metabolopatías, Hospital Universitario Virgen del Rocío, Sevilla, España. 3. Unidad de Neonatología, Hospital Universitario Virgen del Rocío, Sevilla, España. 4. Unidad de Pediatría, Hospital Virgen del Valme, Sevilla, España. 5. Unidad de Inmunología, Hospital Universitario Virgen del Rocío, Sevilla, España. 6. Grupo de Neurodesarrollo, Unidad de Pediatría, Hospital Virgen del Rocío, Sevilla, Instituto de Biomedicina de Sevilla (IBiS), Sevilla, España. 7. Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Estocolmo, Suecia; Translational Centre for Regenerative Medicine(TRM), University of Leipzig, Leipzig, Alemania; ImmunoDeficiencyCenter Leipzig at Hospital St Georg gGmbH Leipzig, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Leipzig, Alemania. 8. Sección de Infectología e Inmunodeficiencias, Unidad de Pediatría, Hospital Virgen del Rocío, Sevilla, Instituto de Biomedicina de Sevilla (IBiS), Sevilla, España. Electronic address: olafw.neth.sspa@juntadeandalucia.es.
Abstract
INTRODUCTION: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected infants/children. The measurement of T-cell receptor excision circles (TRECS) and kappa-deleting recombination excision circles (KRECS) can identify neonates with severe T or B-cell lymphopenia. OBJECTIVES: To determine TRECS and KRECS levels from prospectively collected dried blood spot samples (DBS) and to correctly identify severe T and B-cell lymphopenia. MATERIAL AND METHODS: Determination of TRECS and KRECS by multiplex PCR from neonates born in two tertiary hospitals in Seville between February 2014 and May 2014. PCR cut-off levels: TRECS<15 copies/μl, KRECS<10 copies/μl, ACTB (β-actin)>1000 copies/μl. Internal (XLA, ataxia telangiectasia) and external (SCID) controls were included. RESULTS: A total of 1068 out of 1088 neonates (mean GA 39 weeks (38-40) and BW 3238g (2930-3520) were enrolled in the study. Mean (median, min/max) copies/μl, were as follows: TRECS 145 (132, 8/503), KRECS 82 (71, 7/381), and ACTB 2838 (2763, 284/7710). Twenty samples (1.87%) were insufficient. Resampling was needed in one neonate (0.09%), subsequently giving a normal result. When using lower cut-offs (TRECS<8 and KRECS<4 copies/μl), all the samples tested were normal and the internal and external controls were correctly identified. CONCLUSION: This is the first prospective pilot study in Spain using TRECS/KRECS/ACTB-assay, describing the experience and applicability of this method to identify severe lymphopenias. The ideal cut-off remains to be established in our population. Quality of sampling, storage and preparation need to be further improved.
INTRODUCTION: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected infants/children. The measurement of T-cell receptor excision circles (TRECS) and kappa-deleting recombination excision circles (KRECS) can identify neonates with severe T or B-cell lymphopenia. OBJECTIVES: To determine TRECS and KRECS levels from prospectively collected dried blood spot samples (DBS) and to correctly identify severe T and B-cell lymphopenia. MATERIAL AND METHODS: Determination of TRECS and KRECS by multiplex PCR from neonates born in two tertiary hospitals in Seville between February 2014 and May 2014. PCR cut-off levels: TRECS<15 copies/μl, KRECS<10 copies/μl, ACTB (β-actin)>1000 copies/μl. Internal (XLA, ataxia telangiectasia) and external (SCID) controls were included. RESULTS: A total of 1068 out of 1088 neonates (mean GA 39 weeks (38-40) and BW 3238g (2930-3520) were enrolled in the study. Mean (median, min/max) copies/μl, were as follows: TRECS 145 (132, 8/503), KRECS 82 (71, 7/381), and ACTB 2838 (2763, 284/7710). Twenty samples (1.87%) were insufficient. Resampling was needed in one neonate (0.09%), subsequently giving a normal result. When using lower cut-offs (TRECS<8 and KRECS<4 copies/μl), all the samples tested were normal and the internal and external controls were correctly identified. CONCLUSION: This is the first prospective pilot study in Spain using TRECS/KRECS/ACTB-assay, describing the experience and applicability of this method to identify severe lymphopenias. The ideal cut-off remains to be established in our population. Quality of sampling, storage and preparation need to be further improved.
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