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.


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.

Keywords: KRECS; New born screening; TRECS; severe lymphopenias.

Publication types

  • Multicenter Study
  • Observational Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • B-Lymphocytes / immunology*
  • Birth Weight
  • Case-Control Studies
  • Dried Blood Spot Testing* / standards
  • False Positive Reactions
  • Female
  • Genetic Markers
  • Gestational Age
  • Humans
  • Immunologic Deficiency Syndromes / blood
  • Immunologic Deficiency Syndromes / diagnosis*
  • Immunologic Deficiency Syndromes / genetics
  • Immunologic Deficiency Syndromes / immunology
  • Infant, Low Birth Weight / blood
  • Infant, Low Birth Weight / immunology
  • Infant, Newborn
  • Infant, Premature / blood
  • Infant, Premature / immunology
  • Longitudinal Studies
  • Male
  • Multiplex Polymerase Chain Reaction* / standards
  • Neonatal Screening / methods*
  • Neonatal Screening / standards
  • Predictive Value of Tests
  • Prospective Studies
  • Real-Time Polymerase Chain Reaction* / standards
  • Reproducibility of Results
  • Risk Factors
  • Severity of Illness Index
  • Spain
  • T-Lymphocytes / immunology*


  • Genetic Markers