Beatriz de Felipe1, Peter Olbrich1, José Manuel Lucenas2, Carmen Delgado-Pecellin3, Antonio Pavon-Delgado4, Josefina Marquez5, Carmen Salamanca6, Pere Soler-Palacin7, Luis Ignacio Gonzalez-Granado8, Laura Ferreras Antolin9, Stephan Borte10,11,12, Olaf Neth1. 1. Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, 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), Barcelona, 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, Málaga, Spain. 10. Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 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.
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 DBS were tested. Re-punch was needed in 77 samples (1.5%) due to insufficient β-actin (<700 copies/punch). Pre-term neonates (GA<37 weeks) and neonates with a BW<2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeat positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n = 1; TRECs 1/KRECs 4); extreme pre-maturity (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. Pre-maturity and low BW is associated with lower TREC and KREC levels. Extreme pre-maturity and maternal immune suppressive therapy may be a cause for false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved.
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 DBS were tested. Re-punch was needed in 77 samples (1.5%) due to insufficient β-actin (<700 copies/punch). Pre-term neonates (GA<37 weeks) and neonates with a BW<2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeat positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n = 1; TRECs 1/KRECs 4); extreme pre-maturity (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. Pre-maturity and low BW is associated with lower TREC and KREC levels. Extreme pre-maturity and maternal immune suppressive therapy may be a cause for false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved.
Authors: Maartje Blom; Rolf H Zetterström; Asbjørg Stray-Pedersen; Kimberly Gilmour; Andrew R Gennery; Jennifer M Puck; Mirjam van der Burg Journal: J Allergy Clin Immunol Date: 2021-09-16 Impact factor: 14.290
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