Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville
Beatriz de Felipe1, Peter Olbrich1, José Manuel Lucenas2
1Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
Insights
Early diagnosis of primary immunodeficiency is improved by T-cell-receptor-excision circles (TRECs) and kappa-deleting-recombination-excision circles (KRECs) testing. Preterm birth and low birth weight are associated with lower TRECs and KRECs levels.
Area of Science:
- Neonatal screening
- Immunology
- Genetics
Background:
- Early diagnosis of primary immunodeficiencies like SCID and XLA is crucial for improving child outcomes.
- TRECs and KRECs from DBS can identify neonates with severe T- and/or B-lymphopenia.
- Prospective data on the impact of gestational age and birth weight on TRECs and KRECs are lacking.
Purpose of the Study:
- To investigate the impact of gestational age and birth weight on TRECs and KRECs levels in neonates.
- To evaluate the TRECs-KRECS-β-actin-Assay for identifying T- and B-cell lymphopenias.
Main Methods:
- TRECs and KRECs were determined using a triplex RT-PCR assay from prospectively collected DBS.
- Cut-off levels were established for TRECs, KRECs, and β-actin.
- Internal and external quality controls were included.
Main Results:
- 5160 DBS were tested; 1.5% required re-punching due to insufficient β-actin.
- Preterm neonates and those with low birth weight showed significantly lower TRECs and KRECs levels (p < 0.001).
- Five neonates were recalled for repeat positive results, including cases of extreme prematurity and maternal azathioprine exposure.
Conclusions:
- The TRECS-KRECS-β-actin-Assay effectively identifies T- and B-cell lymphopenias.
- Pre-maturity and low birth weight are associated with reduced TREC and KREC levels.
- Extreme pre-maturity and maternal immune suppressive therapy may lead to false positive results; improved DBS handling is recommended.
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.
