Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville.
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.
Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
Beatriz
B
de Felipe
Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
Peter
P
Olbrich
Unidad de Inmunología, Hospital Universitario Virgen del Rocío, Sevilla, Spain.
José Manuel
JM
Lucenas
Unidad de Metabolopatías, Hospital Universitario Virgen del Rocío, Sevilla, Spain.
Carmen
C
Delgado-Pecellin
Unidad de Neonatología, Hospital Universitario Virgen del Rocío, Sevilla, Spain.
Antonio
A
Pavon-Delgado
Unidad de Pediatría, Hospital Virgen de Valme, Sevilla, Spain.
Josefina
J
Marquez
Unidad de Neonatología, Hospital Universitario Virgen de Macarena, Sevilla, Spain.
Carmen
C
Salamanca
Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron, Institut de Recerca Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
Pere
P
Soler-Palacin
Division of Immune Deficiency, Department of Pediatrics, Hospital 12 de Octubre, Madrid, Spain.
Luis Ignacio
LI
Gonzalez-Granado
Unidad de Enfermedades Infecciosas e Inmunodeficiencias, UGP de Pediatría. Hospital Materno-Infantil, Hospital Regional Universitario de Málaga, Málaga, Spain.
Laura Ferreras
LF
Antolin
Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.|Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany.|ImmunoDeficiencyCenter Leipzig at Hospital St Georg gGmbH Leipzig, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Leipzig, Germany.
Stephan
S
Borte
Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
Olaf
O
Neth
D016477:Artifacts; D001402:B-Lymphocytes / Q000276:immunology*; D001724:Birth Weight; D016022:Case-Control Studies; D059788:Dried Blood Spot Testing / Q000592:standards; D005189:False Positive Reactions; D005260:Female; D005819:Genetic Markers; D005865:Gestational Age; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D007230:Infant, Low Birth Weight / Q000097:blood / Q000276:immunology; D007231:Infant, Newborn; D007234:Infant, Premature / Q000097:blood / Q000276:immunology; D008137:Longitudinal Studies; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000592:standards; D015997:Neonatal Screening / Q000379:methods* / Q000592:standards; D011237:Predictive Value of Tests; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D015203:Reproducibility of Results; D012307:Risk Factors; D012720:Severity of Illness Index; D013030:Spain; D013601:T-Lymphocytes / Q000276:immunology*
D005819:Genetic Markers
KRECS; New born screening; TRECS; severe lymphopenias