ABSTRACT
RESUMO Objetivo: Validar a quantificação de T-cell receptor excision circles (TRECs) e kappa-deleting recombination circles (KRECs) por reação em cadeia de polimerase (polymerase chain reaction, PCR) em tempo real (qRT-PCR), para triagem neonatal de imunodeficiências primárias que cursam com defeitos nas células T e/ou B no Brasil. Métodos: Amostras de sangue de recém-nascidos (RN) e controles foram coletadas em papel-filtro. O DNA foi extraído e os TRECs e KRECs foram quantificados por reação duplex de qRT-PCR. O valor de corte foi determinado pela análise de Receiver Operating Characteristics Curve, utilizando-se o programa Statistical Package for the Social Sciences (SSPS) (IBM®, Armonk, NY, EUA). Resultados: 6.881 amostras de RN foram analisadas quanto à concentração de TRECs e KRECs. Os valores de TRECs variaram entre 1 e 1.006 TRECs/µL, com média e mediana de 160 e 139 TRECs/µL, respectivamente. Três amostras de pacientes diagnosticados com imunodeficiência grave combinada (severe combined immunodeficiency, SCID) apresentaram valores de TRECs abaixo de 4/µL e um paciente com Síndrome de DiGeorge apresentou TRECs indetectáveis. Os valores de KRECs encontraram-se entre 10 e 1.097 KRECs/µL, com média e mediana de 130 e 108 KRECs/µL, e quatro pacientes com diagnóstico de agamaglobulinemia tiveram resultados abaixo de 4 KRECs/µL. Os valores de corte encontrados foram 15 TRECs/µL e 14 KRECs/µL, e foram estabelecidos de acordo com a análise da Receiver Operating Characteristics Curve, com sensibilidade de 100% para detecção de SCID e agamaglobulinemia, respectivamente. Conclusões: A quantificação de TRECs e KRECs foi capaz de diagnosticar crianças com linfopenias T e/ou B em nosso estudo, validando a técnica e dando o primeiro passo para a implementação da triagem neonatal em grande escala no Brasil.
ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.
Subject(s)
Humans , Infant, Newborn , Infant , Neonatal Screening/methods , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/blood , Brazil , DNA/analysis , Receptors, Antigen, B-Cell/genetics , Pilot Projects , Cross-Sectional Studies , Severe Combined Immunodeficiency/genetics , Real-Time Polymerase Chain ReactionABSTRACT
Primary immunodeficiency disorders (PIDs) are a heterogeneous group of inherited disorders that affect different components of the immune system. There are more than 150 different disorders which have been described till date. Despite major advances in the molecular characterization of PIDs over the last 20 years, many patients remain undiagnosed or are diagnosed too late with severe consequences. Recognizing different clinical manifestations of PID is the first most important step. It should be followed by use of appropriate diagnostic tools from a vast number of investigations available. This review will focus on important presenting features of PID and laboratory approach for diagnosis of suspected cases of PID.